NKp46 BINDING PROTEINS

ABSTRACT

Multispecific proteins that bind and specifically redirect NK cells to lyse a target cell of interest are provided without non-specific activation of NK cells in absence of target cells. The proteins have utility in the treatment of disease, notably cancer or infectious disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/321,650, filed Dec. 22, 2016, which is a U.S. National PhaseApplication of Int'l Appl. No. PCT/EP2015/064063, filed Jun. 23, 2015,which claims priority to U.S. Provisional Application No. 62/108,088,filed Jan. 27, 2015, and U.S. Provisional Application No. 62/017,886,filed Jun. 27, 2014, each of which is incorporated herein by reference.

REFERENCE TO THE SEQUENCE LISTING

This application includes as part of its disclosure a biologicalsequence listing which is being concurrently submitted through EFS-Web.Said biological sequence listing is contained in a file named“11562150001202.txt” which was created on Nov. 7, 2019, and has a sizeof 310,458 bytes, and is hereby incorporated by reference in itsentirety.

FIELD OF THE INVENTION

Multispecific proteins that bind and specifically redirect NK cells tolyse a target cell of interest are provided without non-specificactivation of NK cells in absence of target cells. The proteins haveutility in the treatment of disease, notably cancer or infectiousdisease.

BACKGROUND

Bispecific antibodies binding two different epitopes, offeropportunities for increasing specificity, broadening potency, andutilizing novel mechanisms of action that cannot be achieved with atraditional monoclonal antibody. A variety of formats for bispecificantibodies that bind to two targets simultaneously have been reported.Cross-linking two different receptors using a bispecific antibody toinhibit a signaling pathway has shown utility in a number ofapplications (see, e.g., Jackman, et al., (2010) J. Biol. Chem.285:20850-20859). Bispecific antibodies have also been used toneutralize two different receptors. In other approaches, bispecificantibodies have been used to recruit immune effector cells, where T-cellactivation is achieved in proximity to tumor cells by the bispecificantibody which binds receptors simultaneously on the two different celltypes (see Baeuerle, P. A., et al, (2009) Cancer Res 69(12):4941-4).Approaches developed to date have primarily involved bispecificantibodies that link the CD3 complex on T cells to a tumor-associatedantigen. However in other examples, bispecific antibodies having one armwhich binds CD16 (FcγRIIIa) and another which bound to an antigen ofinterest such as CD19 have been developed (see Kellner et al. (2011)Cancer Lett. 303(2): 128-139).

Natural killer (NK) cells are a subpopulation of lymphocytes that areinvolved in non-conventional immunity. NK cells provide an efficientimmunosurveillance mechanism by which undesired cells such as tumor orvirally-infected cells can be eliminated. Characteristics and biologicalproperties of NK cells include the expression of surface antigensincluding CD16, CD56 and/or CD57, the absence of the alpha/beta orgamma/delta TCR complex on the cell surface; the ability to bind to andkill cells that fail to express “self” MHC/HLA antigens by theactivation of specific cytolytic enzymes, the ability to kill tumorcells or other diseased cells that express a ligand for NK activatingreceptors, and the ability to release protein molecules called cytokinesthat stimulate or inhibit the immune response.

NK cell activity is regulated by a complex mechanism that involves bothactivating and inhibitory signals. Several distinct NK cell receptorshave been identified that play an important role in the NK cell mediatedrecognition and killing of HLA Class I deficient target cells. Onereceptor, although not specific to NK cells, is FcγR3a (CD16) which isresponsible for NK cell mediated ADCC. Another NK cell receptor isNKp46, a member of the Ig superfamily. It is specific to NK cells andits cross-linking, induced by specific mAbs, leads to a strong NK cellactivation resulting in increased intracellular Ca⁺⁺ levels, triggeringof cytotoxicity, and lymphokine release. International patentpublication number WO2005/105858 (Innate Pharma) discloses use ofmonospecific full-length IgG anti-NKp46 antibodies that bind Fcγreceptors for treating hematological malignancies that are Fcγ-positive.Fc gamma receptors on tumor cells (e.g. B cell malignancies) wereproposed to interact with the Fc domain of the anti-NKp46 antibodieswhich bound NK cells, such that the activated NK cells are brought intoclose proximity with their target cells via the two reactive portions ofthe antibody (e.g. the antigen-recognizing domain and the Fc domain),thereby enhancing the efficiency of the treatment.

To date, no NK cell specific bispecific antibodies have been developed.The depleting agents that recruit NK cytotoxicity such as anti-tumorantibodies are typically full-length IgG1 that mediate ADCC via CD16.Despite the existence of a variety of formats for bispecific antibodies,there remains a need in the art for proteins with new and well-definedmechanisms of action that can provide benefits over and can be used inaddition to full-length antibodies.

SUMMARY OF THE INVENTION

The present invention arises from the discovery of functionalmulti-specific proteins (e.g. a polypeptide, a single chain protein, amulti-chain protein, including but not limited to antibody-based proteinformats) that binds NKp46 on NK cells and to an antigen of interest on atarget cell, and is capable of redirecting NK cells to lyse a targetcell that expresses the antigen of interest, e.g. a cell thatcontributes to disease.

Advantageously, in on embodiment, the presence of NK cells and targetcells, the multi-specific protein can bind (i) to antigen of interest ontarget cells and (ii) to NKp46 on NK cells, and, when bound to bothantigen of interest on target cells and NKp46, can induce signaling inand/or activation of the NK cells through NKp46 (the protein acts as anNKp46 agonist), thereby promoting activation of NK cells and/or lysis oftarget cells, notably via the activating signal transmitted by NKp46. Inspecific advantageous embodiments, the multi-specific protein binds toNKp46 in monovalent fashion and, when bound to both antigen of intereston target cells and NKp46, induces signaling in the NK cells throughNKp46. In one embodiment, the protein comprises a first antigen bindingdomain and a second antigen binding domain, wherein one of the first orsecond antigen binding domains binds to a human NKp46 polypeptide andthe other of the first or second antigen binding domains binds anantigen of interest expressed on a target cell.

The multi-specific protein does not, however, substantially induce NKp46signaling (and/or NK activation that results therefrom) in NK cells whenthe protein is not bound to the antigen of interest on target cells(e.g. in the absence of antigen of interest and/or target cells). Bylacking agonist activity at NKp46 (NK cell activation is notsubstantially induced as a result of binding to NKp46) in the absence oftarget cells the multi-specific proteins can avoid unwanted NK cellactivation (e.g. other than at the site of disease). In one embodiment,the bispecific protein binds more strongly (has a greater bindingaffinity) for the antigen of interest (e.g. a cancer antigen) than forNKp46.

In view of the NK-cell selective expression pattern of human NKp46, themulti-specific proteins can direct an immune effector response (e.g.,cytotoxic response) toward a target cell that is substantially limitedto NK cells (e.g., NKp46-expressing cells). Furthermore, becauseFcγRIIIa (CD16) is not present on all NK cells, conventional therapeuticantibodies (e.g. of human isotypes IgG1) designed to exertantibody-dependent cellular toxicity (ADCC) via FcγRIIIa may notmobilize all NK cells; the present proteins on the other hand enable allNK cells to be solicited via NKp46. Because the proteins of theinvention promote lysis of target cells via the activating signaltransmitted by NKp46 and not FcγRs, proteins of the invention cantherefore also be used advantageously in combination with therapeuticagents such as antibodies that induce ADCC via FcγRIIIa (CD16) therebytargeting two separate NK cell cytotoxicity pathways.

In one aspect of any embodiment herein, a multi-specific proteindescribed herein can for example be characterized by:

(a) agonist activity at NKp46, when incubated in the presence ofNKp46-expressing NK cells and target cells; and

(b) lack of agonist activity at NKp46 when incubated with NK cells, e.g.NKp46-expressing NK cells, in the absence of target cells. Optionally,the NK cells are purified NK cells.

Determining whether a protein has agonist activity at NKp46 whenincubated in the presence of NKp46-expressing cells and target cells canfor example be evaluated by incubating the protein together with: (a)NKp46-expressing (e.g., NK cells or reporter cells), and (b) targetcells that do not, in the absence of the multi-specific protein, induceNKp46 signaling in the reporter cells, and assessing whether the proteincauses NKp46 signaling, NK cell activation and/or NK cytotoxicity towardthe target cell. In one embodiment, assessing whether the protein causesNKp46 signaling by measuring a change in a NKp46 signaling pathway, e.g.by monitoring phosphorylation. In one embodiment, reporter cells areused with are designed to produce a detectable signal if NKp46 signalingis triggered.

Determining whether a protein lacks agonist activity when incubated withNK cells in the absence of target cells can for example be evaluated byincubating the protein together with purified NKp46-expressing NK cells.If the protein does not cause NK cell activation (e.g. ofNKp46-expressing NK cells) the protein lacks agonist activity at NKp46.In another embodiment, if the protein does not cause NKp46 signaling theprotein lacks agonist activity at NKp46.

In one aspect of any embodiment herein, a multi-specific proteindescribed herein can for example be characterized by:

(a) ability to activate NKp46-expressing NK cells, when incubated withNKp46-expressing NK cells and target cells; and

(b) lack of ability to activate NKp46-expressing NK cells when incubatedwith NKp46-expressing NK cells, in the absence of target cells.Optionally, the NK cells are purified NK cells.

In one aspect of any embodiment herein, a multi-specific proteindescribed herein can for example be characterized by:

(a) ability to induce NKp46-expressing NK cells to lyse target cells,when incubated with NKp46-expressing NK cells and target cells; and

(b) lack of ability to activate NKp46-expressing NK cells, whenincubated with NKp46-expressing NK cells, in the absence of target cells(e.g., NKp46-expressing NK cells alone). Optionally, the NK cells arepurified NK cells.

In one aspect of any embodiment herein, a multi-specific proteindescribed herein can for example be characterized by:

(a) ability to activate NKp46-expressing NK cells and/or mediate NK cellcytotoxicity, when incubated with NKp46-expressing NK cells and targetcells; and

(b) lack of ability to activate NKp46-negative, CD16-positive(NKp46+CD16-) NK cells and/or mediate NK cell cytotoxicity, whenincubated with NKp46-CD16+NK cells and target cells. Optionally, the NKcells are purified NK cells.

In one embodiment, a multi-specific protein has reduced (or lacks)binding to a human Fcγ receptor (e.g. CD16). For example, amulti-specific protein may lack an Fc domain.

In one embodiment, provided are multi-specific protein formats adaptedfor use in a NKp46-based NK cell engager, including antibody-basedformats comprising antigen binding domain(s) and/or constant regiondomain(s) from immunoglobulins. By combining the NK-selective expressionof NKp46 with multi-specific (e.g. bispecific) antibody formats in whichthe multi-specific proteins have reduced (or lack) binding to human Fcγreceptor but maintain at least part of an Fc domain, the inventorsprovide multi-specific antibody formats with favorable pharmacology dueto at least partial FcRn binding and that direct NK cell cytotoxicity toa target of interest, without activating inhibitory Fcγ receptors norblocking activating Fcγ receptors on NK cells (which could reduceefficacy of NK cells) and without triggering inhibitory and/oractivatory Fcγ receptors on other immune cells (e.g. CD16 onmonocyte-derived macrophages) which could lead to unwantedimmunosuppressive effects or unwanted toxicity (e.g. cytokine mediatedtoxicity) and reduced specificity of the overall multi-specific protein,and/or to other unwanted effects such as pro-tumoral effects mediated byFcγ receptor-expressing cells.

In another aspect of any embodiment herein, a multi-specific proteindescribed herein can be characterized by lack of agonist activity atNKp46 when incubated with NK cells in the presence of Fcγreceptor-expressing cells (e.g., Fcγ receptor-expressing lymphocytes),and in the absence of target cells (e.g. cells expressing the antigen ofinterest). In one aspect, a multi-specific protein described herein canbe characterized by lack of ability to activate NKp46-expressing NKcells when incubated with NKp46-expressing NK cells in the presence ofFcγ receptor-expressing cells (e.g., Fcγ receptor-expressinglymphocytes, Fcγ receptor-expressing NK cells), and in the absence oftarget cells (e.g. cells expressing the antigen of interest).

In one embodiment, a multi-specific protein can for example becharacterized by:

(a) agonist activity at NKp46, when incubated in the presence ofNKp46-expressing cells (e.g. NK cells) and target cells; and

(b) lack of agonist activity at NKp46 when incubated with NK cells inthe presence of Fcγ receptor-expressing cells (e.g., Fcγreceptor-expressing lymphocytes), and in the absence of target cells(cells expressing the antigen of interest).

In one embodiment, a multi-specific protein can for example becharacterized by:

(a) ability to activate NKp46-expressing NK cells, when incubated in thepresence of NKp46-expressing cells (e.g. NK cells) and target cells; and

(b) lack of ability to activate NKp46-expressing NK cells, whenincubated with NK cells in the presence of Fcγ receptor-expressing cells(e.g., Fcγ receptor-expressing lymphocytes), and in the absence oftarget cells (cells expressing the antigen of interest).

Determining whether a protein lacks agonist activity when incubated withNK cells in the presence of Fcγ receptor-expressing cells and in theabsence of target cells can for example be evaluated by incubating theprotein together with NK cells in the presence of Fcγreceptor-expressing lymphocytes (e.g. by incubating the protein withPBMC), but without target cells.

In one embodiment, provided is a method for identifying, testing and/orproducing a multispecific protein that binds NKp46 on an NK cell and anantigen of interest expressed by a target cell, the method comprising:

(a) assessing whether the multispecific protein has agonist activity atNKp46, when incubated in the presence of NKp46-expressing cells (e.g. NKcells) and target cells; and

(b) assessing whether the multispecific protein has agonist activity atNKp46 when incubated with NK cells (optionally further in the presenceof Fcγ receptor-expressing cells), in the absence of target cells.

Optionally, the NK cells are purified NK cells.

In one embodiment, provided is a method for identifying, testing and/orproducing a multispecific protein, the method comprising providing aplurality of multispecific proteins protein that bind NKp46 on an NKcell and an antigen of interest expressed by a target cell:

(a) assessing each multispecific protein for agonist activity at NKp46,when incubated in the presence of NKp46-expressing cells (e.g. NK cells)and target cells;

(b) assessing each multispecific protein for agonist activity at NKp46when incubated with NK cells (optionally further in the presence of Fcγreceptor-expressing cells), in the absence of target cells; and

(c) selecting a multispecific protein (e.g. for use as a medicament, forfurther evaluation, for further production, etc.) if the multispecificprotein:

a. has agonist activity at NKp46, when incubated in the presence ofNKp46-expressing cells (e.g. NK cells) and target cells, and b. lacksagonist activity at NKp46 when incubated with NK cells (optionallyfurther with Fcγ receptor-expressing cells), in the absence of targetcells. In any of the embodiments, agonist activity (or lack thereof) canbe characterized by the ability (or lack thereof) to activateNKp46-expressing NK cells, e.g. as assessed by expression of NK cellactivation markers, the induction of NK cytotoxicity, or other suitableassays of increased NK cell activity.

Further provided are certain epitopes on NKp46 are well suited fortargeting with NKp46 binding moieties that lead to bispecific proteinswith advantageous properties, notably high efficacy in directed NK cellsto lyse target cells (e.g. via NKp46-mediated signaling). Provided alsoare CDRs of different anti-NKp46 antibodies suitable for use inconstruction of efficient multi-specific proteins, and amino acidsequences of exemplary multi-specific proteins.

In one embodiment, provided is a multispecific protein (e.g.polypeptide, a non-antibody polypeptide, an antibody) comprising: (a) afirst antigen binding domain; and (b) a second antigen binding domain,wherein one of the first antigen binding domains binds NKp46 and theother binds an antigen of interest on a target cell (other than NKp46),wherein the multispecific protein is capable of directingNKp46-expressing NK cells to lyse said target cell. In one embodiment,the protein comprises at least a portion of a human Fc domain, e.g. anFc domain that is bound by FcRn, optionally wherein the multispecificantibody is designed to have decreased or substantially lack FcγRbinding; in one embodiment, the Fc domain is interposed between the twoABDs (one ABD is placed N-terminal and the other is C-terminal to the Fcdomain).

In one aspect, the multispecific protein is a single chain protein. Inone aspect, the multispecific protein comprises two or more polypeptidechains, i.e. a multi-chain polypeptide. For example, the multispecificprotein or multi-chain protein is a dimer, trimer or tetramer.

An antigen binding domain positioned on a polypeptide chain can bindsits target (i.e., NKp46 or an antigen of interest) as such or canoptionally binds its target together with a complementary protein domain(antigen binding domain) positioned on a different polypeptide chain,wherein the two polypeptide chains associate to form a multimer (e.g.dimer, trimer, etc.).

In one aspect, the multispecific protein binds an NKp46 polypeptide(e.g. of the surface of a NK cell) in monovalent fashion. In one aspect,the protein binds the antigen of interest monovalent fashion.

In one aspect, the protein (and/or the antigen binding domain thereofthat binds NKp46) competes for binding to a NKp46 polypeptide with anyone or any combination of monoclonal antibodies NKp46-1, -2, -3, -4, -6or -9 or the Anti-CD19-F2-Anti-NKp46-1, -2, -3,-4, -6 or -9 bispecificantibodies. In one embodiment, the antigen binding domain that bindsNKp46 binds an epitope on an NKp46 polypeptide of SEQ ID NO:1 comprisingone, two, three or more residues selected from the residues bound by anyone or combination of antibodies NKp46-1, -2, -3, -4, -6 or -9 or theAnti-CD19-F2-Anti-NKp46-1, -2, -3, -4, -6 or -9 bispecific antibodies.In one embodiment the multispecific protein is capable of binding tohuman neonatal Fc receptor (FcRn). In one embodiment the multispecificprotein has decreased or abolished binding to a human and/or non-humanprimate (e.g. cynomolgus monkey) Fcγ receptor, e.g., compared to a fulllength wild type human IgG1 antibody. In one embodiment themultispecific protein has decreased (e.g. partial or complete loss of)antibody dependent cytotoxicity (ADCC), complement dependentcytotoxicity (CDC), antibody dependent cellular phagocytosis (ADCP),FcR-mediated cellular activation (e.g. cytokine release through FcRcross-linking), and/or FcR-mediated platelet activation/depletionmediated by NKp46-negative effector cells.

In another embodiment, provided is a monomeric or multimericmultispecific single or multi-chain protein comprising: (a) a firstantigen binding domain (ABD); (b) a second antigen binding domain,wherein one of the first or second antigen binding domains binds toNKp46 and the other binds to an antigen of interest on a target cell(other than NKp46); and (c) at least a portion of a human Fc domain,wherein the Fc domain is capable of binding to human neonatal Fcreceptor (FcRn) and has decreased binding to a human Fcγ receptor, e.g.,compared to a full length wild type human IgG1 antibody. In oneembodiment the multispecific protein has decreased (e.g. partial orcomplete loss of) antibody dependent cytotoxicity (ADCC), complementdependent cytotoxicity (CDC), antibody dependent cellular phagocytosis(ADCP), FcR-mediated cellular activation (e.g. cytokine release throughFcR cross-linking), and/or FcR-mediated platelet activation/depletionmediated by NKp46-negative effector cells. In one embodiment themultispecific protein is monomeric. In one embodiment the multispecificFc-derived protein is a dimer, e.g. a heterodimer. In one embodiment,the monomeric or dimeric protein comprises a protein with a domainstructure in which an Fc domain is interposed between the first antigenbinding domain (ABD) that binds to NKp46 and the second antigen bindingdomain that binds an antigen of interest. In one embodiment themultispecific Fc-derived polypeptide is a bispecific antibody.

In one embodiment of any of the protein herein, the antigen bindingdomain that binds to an antigen of interest binds to an antigen (e.g.polypeptide) expressed by a target cell which sought to be lysed by anNK cell. Optionally such an antigen is expressed by a cancer cell, avirally infected cell, or a cell that contributes to an autoimmunity orinflammatory disease.

In one embodiment, the multispecific protein binds NKp46 in monovalentfashion. In one embodiment, the multispecific protein binds to theantigen of interest in monovalent fashion. In one embodiment, themultispecific protein binds both NKp46 and the antigen of interest inmonovalent fashion.

In one embodiment, the first antigen binding domain comprises anantibody heavy chain variable domain and a light chain variable domain.Optionally, both said heavy and light chain variable domains areinvolved in binding interactions with NKp46.

In one embodiment, the second antigen binding domain comprises anantibody heavy chain variable domain and a light chain variable domain.Optionally, both said heavy and light chain variable domains areinvolved in binding interactions with the antigen bound by the secondantigen binding domain.

Optionally, the Fc domain comprises at least a portion of a CH2 domainand at least a portion of a CH3 domain.

In one embodiment, the CH2 domain comprises an amino acid modification,compared to a wild-type CH2 domain. In one embodiment, the CH2modification reduces binding of the bispecific polypeptide to a humanFcγ receptor. In one embodiment, the CH2 domain comprises aN297×mutation (EU numbering as in Kabat), wherein X is any amino acidother than asparagine. In one embodiment, the CH3 domain comprises anamino acid modification, compared to a wild-type CH3 domain.

In one embodiment, the CH2 domain and/or CH3 domains are naturallyoccurring (non-mutated) human CH2 and/or CH3 domains. In one embodiment,the multispecific protein comprises an Fc derived polypeptide lacksN-linked glycosylation or has modified N-linked glycosylation.

In one embodiment, the Fc-derived polypeptide is a monomer.

In one embodiment, the Fc-derived polypeptide is a dimer, optionally ahomodimer or a heterodimer. In one embodiment, the Fc-derivedpolypeptide is a heterotrimer. In one embodiment, the Fc-derivedpolypeptide is a hetero-tetramer.

In one embodiment, the CH3 domain is does not dimerize with anotherFc-derived polypeptide (e.g. does not substantially form a homodimerwith another identical Fc polypeptide but remains as a heterodimer orheterotrimer; does not form a homodimer and remains as a monomer). Inone embodiment, the CH3 domain comprises amino acid mutations (e.g.substitutions) in the CH3 dimer interface to prevent formation ofCH3-CH3 dimers.

Examples of monomeric bispecific protein are shown in FIGS. 1-3 andFIGS. 6A-6C. In one embodiment, provided is a monomeric bispecificprotein comprising: (a) a first antigen binding domain that binds to anantigen of interest; (b) a second antigen binding domain that bindsNKp46; and (c) at least a portion of a human Fc domain, wherein the Fcdomain does not dimerize with another Fc-derived polypeptide (e.g. doesnot dimerize with an identical monomeric bispecific polypeptide). In oneembodiment, the monomeric bispecific protein is capable of binding tohuman FcRn and has decreased binding to a human Fcγ receptor compared toa wild type full length human IgG1 antibody. In one embodiment, themonomeric bispecific protein has decreased binding to a human Fcγreceptor compared to a polypeptide having a full length wild-type humanIgG1 Fc domain but otherwise identical. Optionally, the Fc domaincomprises a CH2 domain and a modified CH3 domain to prevent CH3-CH3dimerization (e.g. does not dimerize via interactions with another CH3domain in an identical monomeric bispecific polypeptide).

In one embodiment, the Fc domain is interposed between the first antigenbinding domain and the second binding domain on the polypeptide chain,e.g., the polypeptide has a domain arrangement: (ABD₁)-CH2-CH3-(ABD₂),or further wherein the polypeptide has a domain arrangement:(ABD₁)-linker-CH2-CH3-linker-(ABD₂); optionally intervening amino acidsequences are present between any protein domains. In one embodiment,ABD₁ is the antigen binding domain that binds an antigen of interest andABD₂ is the antigen binding domain that binds to NKp46

In one aspect of any embodiment, the first antigen binding domain and/orthe second antigen binding domain comprise a heavy and/or light chainvariable domain. In one aspect of any embodiment, the first antigenbinding domain and/or the second antigen binding domain comprise a scFv,optionally where the scFv comprises human framework amino acidsequences.

Optionally the monomeric polypeptide is capable of binding to human FcRnwith intermediate affinity, e.g. binds to FcRn but has decreased bindingto a human FcRn receptor compared to a full length wild type human IgG1antibody; optionally the monomeric polypeptide further has decreasedbinding to a human FcγR (e.g. CD16, CD32A, CD32B and/or CD64) comparedto a full length wild type human IgG1 antibody.

In one embodiment, a heteromultimeric protein or polypeptide is atetrameric antibody made up of two heavy chains comprising variableregions (or 1, 2 or 3 CDRs thereof) derived from two different parentalantibodies, and two light chains comprising variable regions (or 1, 2 or3 CDRs thereof) derived from two different parental antibodies. Such atetramer may comprise (a) two heavy chains each comprising a variableregion, a CH1 domain, hinge and an Fc domain, and (b) two antibody lightchains each comprising a light chain variable region and a CK domain,wherein one heavy chain variable region together with a light chainvariable region binds to NKp46 and the other heavy chain variable regiontogether with a light chain variable region bind an antigen of interest.Optionally the Fc domains are of IgG4 isotype or modified (e.g. with anamino acid substitution or produced in an appropriate host cell) toretain FcRn binding but lack of have decrease FcγR binding. In oneembodiment, provided is a heteromultimeric, e.g. heterodimeric,bispecific protein comprising: (a) a first polypeptide chain comprisinga first variable region (V), fused to a CH1 or CK domain, wherein theV—(CH1/CK) unit is in turn fused to a first terminus (N- or C-teminus)of a human Fc domain (a full Fc domain or a portion thereof); (b) asecond polypeptide chain comprising a first variable region (V) fused toa CH1 or CK domain that is complementary with the CH1 or CK of the firstchain to form a CH1-CK dimer, optionally wherein the V—(CH1/CK) unit isfused to at least a human Fc domain (a full Fc domain or a portionthereof), wherein the two first variable regions form an antigen bindingdomain that binds a first antigen of interest in monovalent fashion, and(c) an antigen binding domain that binds a second antigen (optionallytogether with a complementary antigen binding domain), and optionally asecond CH1 or CK domain, fused to a second terminus (N- or C-terminus)of the Fc domain of the first polypeptide such that the Fc domain isinterposed between the V—(CH1/CK) unit and the antigen binding domainthat binds a second antigen, wherein one of the first and secondantigens is NKp46. Optionally the first and second polypeptide chainsare bound by interchain disulfide bonds, e.g. formed between respectiveCH1 and CK domains. Optionally a V—(CH1/CK) unit is fused to a human Fcdomain directly, or via intervening sequences, e.g. linkers, otherprotein domain(s), etc.

In one embodiment of the above heteromultimeric polypeptide or protein,the polypeptide or protein is a heterodimer, wherein the antigen bindingdomain for a second antigen is an scFv, optionally an scFv that bindsNKp46.

In one embodiment of the above heteromultimeric polypeptide or protein,the polypeptide or protein is a heterotrimer, wherein the antigenbinding domain for a second antigen is an heavy or light chain variableregion, and the heteromultimeric polypeptide or protein furthercomprises a third polypeptide chain comprising a variable region (V)fused to a CH1 or CK domain that is complementary with the CH1 or CK ofthe first chain to form a CH1-CK dimer wherein the variable region thatis the antigen binding domain for a second antigen of the firstpolypeptide and the variable region of the third chain form an antigenbinding domain. The three polypeptide chains formed from the doubledimerization yields a trimer. The CH1 or CK constant region of the thirdpolypeptide is selected to be complementary to the second CH1 or CKconstant region of the first polypeptide chain (but not complementary tothe first CH1/CK of the first polypeptide chain).

In one aspect provided is an isolated heterodimeric polypeptide thatbinds a first and second antigen of interest in monovalent fashion,wherein one of the antigens is NKp46 and the other is an antigen ofinterest, comprising:

(a) a first polypeptide chain comprising, from N- to C-terminus, a firstvariable domain (V), a CH1 of CK constant region, a Fc domain or portionthereof, a second variable domain and third variable domain; and

(b) a second polypeptide chain comprising, from N- to C-terminus, afirst variable domain (V), a CH1 or CK constant region, and optionally aFc domain or portion thereof, wherein the CH1 or CK constant region isselected to be complementary to the CH1 or CK constant region of thefirst polypeptide chain such that the first and second polypeptides forma CH1-CK heterodimer in which the first variable domain of the firstpolypeptide chain and the first variable domain of the secondpolypeptide form an antigen binding domain that binds the first antigenof interest; and wherein a second variable domain and third variabledomain forms an antigen binding domain that binds the second antigen ofinterest. When the second polypeptide chain lacks an Fc domain, thefirst polypeptide chain will comprise an Fc domain modified to preventCH3-CH3 dimerization (e.g., substitutions or tandem CH3 domain).

In one aspect provided is an isolated heterodimeric polypeptide thatbinds a first and second antigen of interest in monovalent fashion,wherein one of the antigens is NKp46 and the other is an antigen ofinterest, comprising:

(a) a first polypeptide chain comprising, from N- to C-terminus, asecond variable domain and third variable domain, a Fc domain or portionthereof, a first variable domain (V), and a CH1 of CK constant region;and

(b) a second polypeptide chain comprising, from N- to C-terminus, afirst variable domain (V), a CH1 or CK constant region, and optionally aFc domain or portion thereof, wherein the CH1 or CK constant region isselected to be complementary to the CH1 or CK constant region of thefirst polypeptide chain such that the first and second polypeptides forma CH1-CK heterodimer in which the first variable domain of the firstpolypeptide chain and the first variable domain of the secondpolypeptide form an antigen binding domain that binds the first antigenof interest; and wherein a second variable domain and third variabledomain forms an antigen binding domain that binds the second antigen ofinterest. When the second polypeptide chain lacks an Fc domain, thefirst polypeptide chain will comprise an Fc domain modified to preventCH3-CH3 dimerization (e.g., substitutions or tandem CH3 domain).

In one embodiment, provided is a trimeric polypeptide that binds a firstand second antigen of interest in monovalent fashion, wherein one of theantigens is NKp46 and the other is an antigen of interest, comprising:

(a) a first polypeptide chain comprising, from N- to C-terminus, a firstvariable domain (V) fused to a first CH1 or CK constant region, an Fcdomain or portion thereof, and a second variable domain (V) fused to asecond CH1 or CK constant region;

(b) a second polypeptide chain comprising, from N- to C-terminus, avariable domain fused to a CH1 or CK constant region selected to becomplementary to the first (but not the second) CH1 or CK constantregion of the first polypeptide chain such that the first and secondpolypeptides form a CH1-CK heterodimer, and optionally an Fc domain orportion thereof; and

(c) a third polypeptide chain comprising, from N- to C-terminus, avariable domain fused to a CH1 or CK constant region, wherein the CH1 orCK constant region is selected to be complementary to the second (butnot the first) variable domain and second CH1 or CK constant region ofthe first polypeptide chain. The first and third polypeptides willtherefore form a CH1-CK heterodimer formed between the CH1 or CKconstant region of the third polypeptide and the second CH1 or CKconstant region of the first polypeptide, but not between the CH1 or CKconstant region of the third polypeptide and the first CH1 or CKconstant region of the first polypeptide. The first, second and thirdpolypeptides form a CH1-CK heterotrimer, and wherein the first variabledomain of the first polypeptide chain and the variable domain of thesecond polypeptide chain form an antigen binding domain specific for afirst antigen of interest, and the second variable domain of the firstpolypeptide chain and the variable domain on the third polypeptide chainform an antigen binding domain specific for a second antigen ofinterest.

In one embodiment, the above heteromultimeric polypeptide or proteincomprises one or more additional polypeptide chains.

In one embodiment, a heteromultimeric polypeptide or protein comprises amonomeric Fc domain (e.g. the second polypeptide does not comprise an Fcdomain), optionally wherein the Fc domain comprises a CH3 domain with anamino acid mutation to prevent CH3-CH3 dimerization or a tandem CH3domain.

In one embodiment, the above heteromultimeric polypeptide or proteincomprises a dimeric Fc domain.

Optionally the heterodimeric polypeptide or protein is capable ofbinding to human FcRn with intermediate affinity, e.g. binds to FcRn buthas decreased binding to a human FcRn receptor compared to a full lengthwild type human IgG1 antibody; optionally the monomeric polypeptidefurther has decreased binding to a human FcγR receptor (e.g. CD16,CD32A, CD32B and/or CD64) compared to a full length wild type human IgG1antibody.

Optionally, the CH1 and/or CK domain are fused via a hinge region to theFc domain. Optionally the hinge, CH2 and/or CH3 comprise an amino acidmodification to reduce or substantially abolish binding to a human Fcγreceptor (e.g. CD16, CD32A, CD32B and/or CD64). Optionally such mutationdecreases (e.g. partial or complete loss of) antibody dependentcytotoxicity (ADCC), complement dependent cytotoxicity (CDC), antibodydependent cellular phagocytosis (ADCP), FcR-mediated cellular activation(e.g. cytokine release through FcR cross-linking), and/or FcR-mediatedplatelet activation/depletion by NKp46-negative cells. Preferably, inany embodiment herein, CH1 and CK domains are of human origin.

In one aspect of any of the embodiments herein, the bispecific proteinbinds more strongly (has a greater binding affinity) for the antigen ofinterest (e.g. a cancer antigen) than for NKp46. Such antibodies willprovide for advantageous pharmacological properties. In one aspect ofany of the embodiments herein, the polypeptide has a Kd for binding(monovalent) to NKp46 of less than 10⁻⁷ M, preferably less than 10⁻⁸ M,or preferably less than 10⁻⁹ M for binding to a NKp46 polypeptide;optionally the polypeptide has a Kd for binding (monovalent) to acancer, viral or bacterial antigen that is less than (i.e. has betterbinding affinity than) the Kd for binding (monovalent) to a NKp46polypeptide. In one aspect of any of the embodiments herein, thepolypeptide has a Kd for binding (monovalent) to NKp46 of between 10⁻⁷ M(100 nanomolar) and 10⁻¹⁰ M (0.1 nanomolar) for binding to a NKp46polypeptide. In one aspect of any of the embodiments herein, thepolypeptide has a Kd for binding (monovalent) to NKp46 of between 10⁻⁸ M(10 nanomolar) and 10⁻¹⁰ M (0.1 nanomolar) for binding to a NKp46polypeptide. In one aspect of any of the embodiments herein, thepolypeptide has a Kd for binding (monovalent) to NKp46 of between 10⁻⁸ M(10 nanomolar) and 10⁻⁹M (1 nanomolar) for binding to a NKp46polypeptide.

In one aspect of any of the embodiments of the invention, the antigenbinding domain that binds NKp46 binds to at least one residue on NKp46corresponding to an amino acid residues bound by any one of monoclonalantibodies NKp46-1, -2, -3, -4, -6 or -9 or the Anti-CD19-Anti-NKp46-1,-2, -3, -4, -6 or -9 bispecific antibodies. In one aspect, the antigenbinding domain that binds NKp46 binds at least 1, 2, 3, 4 or more aminoacids of NKp46 within the epitope bound by any one or combination ofmonoclonal antibodies NKp46-1, -2, -3, -4, -6 or -9 or theAnti-CD19-Anti-NKp46-1, -2, -3, -4, -6 or -9 bispecific antibodies. Inone aspect of any of the embodiments of the invention, the antigenbinding domain that binds NKp46 binds to the same epitope on a NKp46polypeptide as any of monoclonal antibodies NKp46-1, -2, -3, -4, -6 or-9 or the Anti-CD19-Anti-NKp46-1, -2, -3, -4, -6 or -9 bispecificantibodies. In one embodiment, the antigen binding domain that bindsNKp46 binds an epitope on an NKp46 polypeptide of SEQ ID NO:1 comprisingone, two, three or more residues selected from the group of residuesbound by any of antibodies NKp46-1, -2, -3, -4,-6 or -9.

In some embodiments, the protein that binds NKp46 exhibits significantlylower binding for a mutant NKp46 polypeptide in which a residue bound byany of antibodies NKp46-1, -2, -3, -4, -6 or -9 is substituted with adifferent amino acid, compared to a wild-type NKp46 polypeptide of SEQID NO: 1.

In one aspect of any of the embodiments of the invention, the proteinthat binds NKp46 competes for binding to a NKp46 polypeptide with anyone or any combination of monoclonal antibodies NKp46-1, NKp46-2,NKp46-3, NKp46-4, NKp46-6 or NKp46-9, or the Anti-CD19-Anti-NKp46-1, -2,-3, -4, -6 or -9 bispecific antibodies. In one embodiment, the proteinthat binds NKp46 competes for binding to a NKp46 polypeptide with anantibody selected from the group consisting of:

(a) an antibody having respectively a VH and VL region of SEQ ID NOS: 3and 4 (NKp46-1);

(b) an antibody having respectively a VH and VL region of SEQ ID NOS: 5and 6 (NKp46-2);

(c) (a) an antibody having respectively a VH and VL region of SEQ IDNOS: 7 and 8 (NKp46-3);

(d) (a) an antibody having respectively a VH and VL region of SEQ IDNOS: 9 and 10 (NKp46-4);

(e) an antibody having respectively a VH and VL region of SEQ ID NOS:11and 12 (NKp46-6); and

(f) an antibody having respectively a VH and VL region of SEQ ID NOS: 13and 14 (NKp46-9).

In one embodiment, provided is an isolated protein that specificallybinds NKp46 (e.g. a monospecific monoclonal antibody, a multispecificpolypeptide, a bispecific antibody) that competes for binding to a NKp46polypeptide with an antibody selected from the group consisting of:

(a) an antibody having respectively a VH and VL region of SEQ ID NOS: 3and 4 (NKp46-1);

(b) an antibody having respectively a VH and VL region of SEQ ID NOS: 5and 6 (NKp46-2);

(c) (a) an antibody having respectively a VH and VL region of SEQ IDNOS: 7 and 8 (NKp46-3);

(d) (a) an antibody having respectively a VH and VL region of SEQ IDNOS: 9 and 10 (NKp46-4);

(e) an antibody having respectively a VH and VL region of SEQ ID NOS:11and 12 (NKp46-6); and

(f) an antibody having respectively a VH and VL region of SEQ ID NOS: 13and 14 (NKp46-9).

In one aspect of any of the embodiments of the invention, the antigenbinding domain that binds NKp46 comprises the hypervariable regions ofany one of monoclonal antibodies NKp46-1, NKp46-2, NKp46-3, NKp46-4,NKp46-6 or NKp46-9.

In one aspect of any of the embodiments of the invention, the antigenbinding domain that binds NKp46 has a heavy and/or light chain variableregion having one, two or three CDRs of the respective heavy and/orlight chain of an antibody selected from the group consisting ofantibody NKp46-1, NKp46-2, NKp46-3, NKp46-4, NKp46-6 and NKp46-9.

In one aspect, provided is an isolated multispecific protein (amonomeric or multimeric polypeptide) that specifically binds (i) NKp46and (ii) an antigen of interest (other than NKp46), wherein themultispecific protein comprises a monomeric Fc domain comprising anamino acid sequence which is at least 60%, 70%, 80%, 85%, 90%, 95% or98% identical to the sequence of SEQ ID NOS: 2, optionally wherein one,two, three, four, five or more amino acids are substituted by adifferent amino acid, optionally comprising a substitution at 1, 2, 3,4, 5, 6 of residues 121, 136, 165, 175, 177 or 179 of SEQ ID NO: 2. Inone embodiment, an isolated multispecific protein that binds NKp46according to the disclosure comprises or an antigen binding domainthereof comprises heavy chain CDR1, 2 and 3 and light chain CDR 1, 2 and3 of any of the antibodies selected from the group consisting of:

(a) an antibody having respectively a VH and VL region of SEQ ID NOS: 3and 4 (NKp46-1);

(b) an antibody having respectively a VH and VL region of SEQ ID NOS: 5and 6 (NKp46-2);

(c) (a) an antibody having respectively a VH and VL region of SEQ IDNOS: 7 and 8 (NKp46-3);

(d) (a) an antibody having respectively a VH and VL region of SEQ IDNOS: 9 and 10 (NKp46-4);

(e) an antibody having respectively a VH and VL region of SEQ ID NOS:11and 12 (NKp46-6); and

(f) an antibody having respectively a VH and VL region of SEQ ID NOS: 13and 14 (NKp46-9).

In one embodiment, an antibody or antigen binding domain according tothe disclosure that binds NKp46 comprises:

(a) (i) a heavy chain comprising a CDR 1, 2 and 3 of the heavy chainvariable region of NKp46-1 of Table A, and (ii) a light chain comprisinga CDR 1, 2 and 3 of the light chain variable region of NKp46-1 of TableA;

(b) (i) a heavy chain comprising a CDR 1, 2 and 3 of the heavy chainvariable region of NKp46-2 of Table A and (ii) a light chain comprisinga CDR 1, 2 and 3 of the light chain variable region of NKp46-2 of TableA;

(c) (i) a heavy chain comprising a CDR 1, 2 and 3 of the heavy chainvariable region of NKp46-3 of Table A and (ii) a light chain comprisinga CDR 1, 2 and 3 of the light chain variable region of NKp46-3 of TableA;

(d) (i) a heavy chain comprising a CDR 1, 2 and 3 of the heavy chainvariable region of NKp46-4 of Table A and (ii) a light chain comprisinga CDR 1, 2 and 3 of the light chain variable region of NKp46-4 of TableA;

(e) (i) a heavy chain comprising a CDR 1, 2 and 3 of the heavy chainvariable region of NKp46-6 of Table A and (ii) a light chain comprisingCDR 1, 2 and 3 of the light chain variable region of NKp46-6 of Table A;or

(f) (i) a heavy chain comprising a CDR 1, 2 and 3 of the heavy chainvariable region of NKp46-9 of Table A and (ii) a light chain comprisinga CDR 1, 2 and 3 of the light chain variable region of NKp46-9 of TableA.

In one aspect, provided is an isolated polypeptide (a monomeric ormultimeric polypeptide) that specifically binds NKp46 (e.g. amonospecific monoclonal antibody, a multispecific polypeptide, abispecific antibody) that binds the same epitope on NKp46 as an antibodyselected from the group consisting of antibody NKp46-1, NKp46-2,NKp46-3, NKp46-4, NKp46-6 and NKp46-9. The isolated polypeptide may be,for example, a monospecific monoclonal antibody, a multispecificpolypeptide or a bispecific antibody

In one aspect, provided is an isolated polypeptide (a monomeric ormultimeric polypeptide) that specifically binds NKp46 (e.g. amonospecific monoclonal antibody, a multispecific polypeptide, abispecific antibody) comprising:

(a) a heavy chain comprising CDR 1, 2 and 3 of the heavy chain variableregion of SEQ ID NO: 3 and a light chain comprising CDR 1, 2 and 3 ofthe light chain variable region of SEQ ID NO: 4;

(b) a heavy chain comprising CDR 1, 2 and 3 of the heavy chain variableregion of SEQ ID NO: 5 and a light chain comprising CDR 1, 2 and 3 ofthe light chain variable region of SEQ ID NO: 6;

(c) a heavy chain comprising CDR 1, 2 and 3 of the heavy chain variableregion of SEQ ID NO: 7 and a light chain comprising CDR 1, 2 and 3 ofthe light chain variable region of SEQ ID NO: 8;

(d) a heavy chain comprising CDR 1, 2 and 3 of the heavy chain variableregion of SEQ ID NO: 9 and a light chain comprising CDR 1, 2 and 3 ofthe light chain variable region of SEQ ID NO: 10;

(e) a heavy chain comprising CDR 1, 2 and 3 of the heavy chain variableregion of SEQ ID NO: 11 and a light chain comprising CDR 1, 2 and 3 ofthe light chain variable region of SEQ ID NO: 12; or

(f) a heavy chain comprising CDR 1, 2 and 3 of the heavy chain variableregion of SEQ ID NO: 13 and (a light chain comprising CDR 1, 2 and 3 ofthe light chain variable region of SEQ ID NO: 14.

In one aspect, provided is an isolated multispecific heterodimericprotein comprising a first polypeptide chain comprising a first aminoacid sequence which is at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 98%identical to the sequence of a first polypeptide chain of a F1 to F17polypeptides disclosed herein; and a second amino acid sequence which isat least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 98% identical to thesequence of a second polypeptide chain of the respective F1 to F17polypeptide disclosed herein. Optionally any or all of the variableregions or CDRs of the first and second chains are substituted withdifferent variable regions, optionally where variable regions or CDRsare excluded from the sequences that are considered for computingidentity, optionally wherein the anti-NKp46 variable regions or CDRs areincluded for computing identity and the variable regions or CDRs for theantigen binding domain that binds the other antigen are excluded fromthe sequences that are considered for computing identity.

In one aspect, provided is an isolated multispecific heterotrimericprotein comprising a first polypeptide chain comprising a first aminoacid sequence which is at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 98%identical to the sequence of a first polypeptide chain of the F1 to F17polypeptides disclosed herein; a second amino acid sequence which is atleast 50%, 60%, 70%, 80%, 85%, 90%, 95% or 98% identical to the sequenceof a second polypeptide chain of the respective F1 to F17 polypeptidedisclosed herein; and a third amino acid sequence which is at least 50%,60%, 70%, 80%, 85%, 90%, 95% or 98% identical to the sequence of a thirdpolypeptide chain of the respective F1 to F17 polypeptide disclosedherein. Optionally any or all of the variable regions or CDRs of thefirst and second chains are substituted with different variable regions,optionally where variable regions or CDRs are excluded from thesequences that are considered for computing identity, optionally whereinthe anti-NKp46 variable regions or CDRs are included for computingidentity and the variable regions or CDRs for the antigen binding domainthat binds the other antigen are excluded from the sequences that areconsidered for computing identity.

In one embodiment of any of the polypeptides herein, the multispecificpolypeptide is capable of directing NKp46-expressing NK cells to lyse atarget cell of interest (e.g. a target cell expressing an antigen otherthan NKp46).

In one aspect of any of the embodiments herein, provided is arecombinant nucleic acid encoding a first polypeptide chain, and/or asecond polypeptide chain and/or a third polypeptide chain of any of theproteins of the disclosure. In one aspect of any of the embodimentsherein, provided is a recombinant host cell comprising a nucleic acidencoding a first polypeptide chain, and/or a second polypeptide chainand/or a third polypeptide chain of any of the proteins of thedisclosure, optionally wherein the host cell produces a protein of thedisclosure with a yield (final productivity after purification) of atleast 1, 2, 3 or 4 mg/L. Also provided is a kit or set of nucleic acidscomprising a recombinant nucleic acid encoding a first polypeptide chainof the disclosure, a recombinant nucleic acid encoding a secondpolypeptide chain of the disclosure, and, optionally, a recombinantnucleic acid encoding a third polypeptide chain of the disclosure. Alsoprovided are methods of making monomeric, heterodimeric andheterotrimeric proteins of the disclosure.

Any of the methods can further be characterized as comprising any stepdescribed in the application, including notably in the “DetailedDescription of the Invention”). The invention further relates to methodsof identifying, testing and/or making proteins described herein. Theinvention further relates to a multispecific protein obtainable by anyof present methods. The disclosure further relates to pharmaceutical ordiagnostic formulations of the multispecific protein disclosed herein.The disclosure further relates to methods of using the multispecificprotein in methods of treatment or diagnosis.

In one embodiment, the multispecific protein are administered to anindividual having a disease (e.g. cancer, a viral or bacterial disease)in combination with a therapeutically effective amount of anADCC-inducing antibody. The ADCC-inducing antibody can be, for example,an antibody that binds to a cancer antigen, viral antigen or bacterialantigen comprising an Fc domain that is bound by a human Fcγ receptor(e.g. CD16). In some embodiments, the ADCC-inducing antibody comprises anative or modified Fc domain from a human IgG1 or IgG3 isotype antibody.In some embodiments, the ADCC-inducing antibody has enhanced ADCCactivity, e.g. comprising an Fc domain that comprises one or more aminoacid modifications such as amino acid substitutions or hypofucosylation,compared to a native human IgG Fc domain.

These and additional advantageous aspects and features of the inventionmay be further described elsewhere herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows two examples of multispecific polypeptides in which one ofthe antigen binding domains (ABD₁ or ABD₂) specifically binds to NKp46and the other of the ABDs binds to an antigen of interest, wherein thedrawing on the left has tandem scFv and the drawing on the right has twoABD with an Fc domain interposed.

FIG. 2 shows a schematic of an anti-CD19-F1-Anti-NKp46 used in theExamples herein. The star in the CH2 domain indicates an option N297Smutation.

FIG. 3 shows a schematic of an anti-CD19-Anti-NKp46-IgG1-Fcmono. For thescFv tandem construct, the Anti-NKp46 VK domain (C-terminal) is linkedto the CH2 domain (N-terminal) using a linker peptide (RTVA) that mimicsthe regular VK—CK elbow junction.

FIG. 4 shows that Anti-CD19-F1-Anti-CD3 does not cause T/B cellaggregation in the presence of B221 (CD19) or JURKAT (CD3) cell lineswhen separate, but it does cause aggregation of cells when both B221 andJURKAT cells are co-incubated.

FIG. 5 shows Anti-CD19-F1-Anti-CD3 retains binding to FcRn, with a 1:1ratio (1 FcRn for each monomeric Fc) (KD=194 nM), in comparison to achimeric full length antibody having human IgG1 constant regions(KD=15.4 nM) which binds to FcRn with a 2:1 ration (2 FcRn for eachantibody).

FIG. 6A to 6E shows different domain arrangements of bispecificanti-NKp46 proteins produced.

FIG. 7A shows superimposed sensorgrams showing the raw data curves,sample (NKp46) and blank (Buffer), which were used to generate eachsubtracted sensorgrams of FIG. 7B. FIG. 7B shows superimposedsubstracted sensorgrams showing the binding of NKp46 recombinantproteins to the captured bispecific monomeric polypeptide.

FIGS. 8A and 8B show respectively bispecific F1 and F2 antibodies havingNKp46 binding region based on NKp46-1, NKp46-2, NKp46-3 or NKp46-4 areable to direct resting NK cells to their CD19-positive Daudi tumortarget cells, while isotype control antibody did not lead to eliminationof the Daudi cells. Rituximab (RTX) served as positive control of ADCC,where the maximal response obtained with RTX (at 10 μg/ml in this assay)was 21.6% specific lysis.

FIG. 9A shows bispecific antibodies having NKp46 and CD19 bindingregions in an F2 format protein do not activate resting NK cells in theabsence of target cells, however full length anti-NKp46 antibodies aswell as positive control alemtuzumab did activate NK cells. FIG. 9A.FIG. 9B shows that bispecific anti-NKp46×anti-CD19 antibodies (includingeach of the NKp46-1, NKp46-2, NKp46-3 or NKp46-4 binding domains)activated resting NK cells in presence of Daudi target cells, whilefull-length anti-CD19 showed at best only very low activation of NKcells and neither full-length anti-NKp46 antibodies or alemtuzmab showedsubstantial increase in activation beyond what was observed in presenceof NK cells alone. FIG. 9C shows that in the presence of CD19-negativeHUT78 cells, none of the bispecific anti-NKp46×anti-CD19 antibody(including each of the NKp46-1, NKp46-2, NKp46-3 or NKp46-4 variableregions) activated NK cells. However, the full-length anti-NKp46antibodies and alemtuzumab caused detectable activation of NK cells at asimilar level observed in presence of NK cells alone. Isotype controlantibody did not induce activation.

FIGS. 10A and B shows that at low effector:target ratio of 1:1 each ofthe bispecific anti-NKp46×anti-CD19 antibody activated NK cells in thepresence of Daudi cells, and that bispecific anti-NKp46×anti-CD19 werefar more potent than the anti-CD19 antibody as a full-length human IgG1as ADCC inducing antibody.

FIGS. 11A and 11B shows that each NKp46×CD19 bispecific protein (FormatF3, F5 and F6) induced specific lysis of Daudi or B221 cells by humanKHYG-1 CD16-negative hNKp46-positive NK cell line, while rituximab andhuman IgG1 isotype control (IC) antibodies did not.

FIGS. 12A-12B, 13A-13B, 14A-14B, 15A-15B, 16A-16B, 17A-17B show bindingof antibodies to different NKp46 mutants. Antibody NKp46-1 had decreasedbinding to the mutant 2 (FIG. 12B) compared to wild-type NKp46 (FIG.12A), and decreased binding to the supplementary mutant Supp? (FIG. 13B)compared to wild-type NKp46 (FIG. 13A). Antibody NKp46-3 had decreasedbinding to the mutant Supp8 (FIG. 14B) compared to wild-type NKp46 (FIG.14A), and decreased binding to the supplementary mutant 19 (FIG. 15B)compared to wild-type NKp46 (FIG. 15A). Antibody NKp46-4 had decreasedbinding to the mutant 6 (FIG. 16B) compared to wild-type NKp46 (FIG.16A), and decreased binding to the supplementary mutant Supp6 (FIG. 17B)compared to wild-type NKp46 (FIG. 17A).

FIG. 18 shows superimposed sensorgrams showing the binding of Macacafascicularis recombinant FcgRs (upper panels; CyCD64, CyCD32a, CYCD32b,CyCD16) and of human recombinant FcgRs (lower panels; HuCD64, HuCD32a,HuCD32b. HUCD16a) to the immobilized human IgG1 control (grey) andCD19/NKp46-1 bi-specific antibody (black). While full length wild typehuman IgG1 bound to all cynomolgus and human Fey receptors, theCD19/NKp46-1 bi-specific antibodies did not bind to any of thereceptors.

FIG. 19A shows results of purification by SEC of proteins format 6 (F6),compared with DART and BITE. BITE and DART showed a very low productionyield compared to F6 and have a very complex SEC profile. FIG. 19B showsSOS-PAGE after Coomassie staining in the expected SEC fractions (3 and 4for BITE and 4 and 5 for DART), whereas F6 format showed clear andsimple SEC and SOS-PAGE profiles with a major peak (fraction 3)containing the desired bispecific proteins.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used in the specification, “a” or “an” may mean one or more. As usedin the claim(s), when used in conjunction with the word “comprising”,the words “a” or “an” may mean one or more than one.

Where “comprising” is used, this can optionally be replaced by“consisting essentially of”, more optionally by “consisting of”.

As used herein, the term “antigen binding domain” refers to a domaincomprising a three-dimensional structure capable of immunospecificallybinding to an epitope. Thus, in one embodiment, said domain can comprisea hypervariable region, optionally a VH and/or VL domain of an antibodychain, optionally at least a VH domain. In another embodiment, thebinding domain may comprise at least one complementarity determiningregion (CDR) of an antibody chain. In another embodiment, the bindingdomain may comprise a polypeptide domain from a non-immunoglobulinscaffold.

The term “antibody” herein is used in the broadest sense andspecifically includes full-length monoclonal antibodies, polyclonalantibodies, multispecific antibodies (e.g., bispecific antibodies), andantibody fragments and derivatives, so long as they exhibit the desiredbiological activity. Various techniques relevant to the production ofantibodies are provided in, e.g., Harlow, et al., ANT/BODIES: ALABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., (1988). An “antibody fragment” comprises a portion of afull-length antibody, e.g. antigen-binding or variable regions thereof.Examples of antibody fragments include Fab, Fab′, F(ab)₂, F(ab′)₂,F(ab)₃, Fv (typically the VL and VH domains of a single arm of anantibody), single-chain Fv (scFv), dsFv, Fd fragments (typically the VHand CH1 domain), and dAb (typically a VH domain) fragments; VH, VL, VhH,and V-NAR domains; minibodies, diabodies, triabodies, tetrabodies, andkappa bodies (see, e.g., Ill et al., Protein Eng 1997; 10: 949-57);camel IgG; IgNAR; and multispecific antibody fragments formed fromantibody fragments, and one or more isolated CDRs or a functionalparatope, where isolated CDRs or antigen-binding residues orpolypeptides can be associated or linked together so as to form afunctional antibody fragment. Various types of antibody fragments havebeen described or reviewed in, e.g., Holliger and Hudson, Nat Biotechnol2005; 23, 1126-1136; WO2005040219, and published U.S. PatentApplications 20050238646 and 20020161201.

The term “antibody derivative”, as used herein, comprises a full-lengthantibody or a fragment of an antibody, e.g. comprising at leastantigen-binding or variable regions thereof, wherein one or more of theamino acids are chemically modified, e.g., by alkylation, PEGylation,acylation, ester formation or amide formation or the like. Thisincludes, but is not limited to, PEGylated antibodies,cysteine-PEGylated antibodies, and variants thereof.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody that are responsible for antigen binding.The hypervariable region generally comprises amino acid residues from a“complementarity-determining region” or “CDR” (e.g. residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light-chain variable domain and 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy-chain variable domain;Kabat et al. 1991) and/or those residues from a “hypervariable loop”(e.g. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light-chainvariable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in theheavy-chain variable domain; Chothia and Lesk, J. Mol. Biol 1987;196:901-917). Typically, the numbering of amino acid residues in thisregion is performed by the method described in Kabat et al., supra.Phrases such as “Kabat position”, “variable domain residue numbering asin Kabat” and “according to Kabat” herein refer to this numbering systemfor heavy chain variable domains or light chain variable domains. Usingthe Kabat numbering system, the actual linear amino acid sequence of apeptide may contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or CDR of the variable domain.For example, a heavy chain variable domain may include a single aminoacid insert (residue 52a according to Kabat) after residue 52 of CDR H2and inserted residues (e.g. residues 82a, 82b, and 82c, etc. accordingto Kabat) after heavy chain FR residue 82. The Kabat numbering ofresidues may be determined for a given antibody by alignment at regionsof homology of the sequence of the antibody with a “standard” Kabatnumbered sequence.

By “framework” or “FR” residues as used herein is meant the region of anantibody variable domain exclusive of those regions defined as CDRs.Each antibody variable domain framework can be further subdivided intothe contiguous regions separated by the CDRs (FR1, FR2, FR3 and FR4).

By “constant region” as defined herein is meant an antibody-derivedconstant region that is encoded by one of the light or heavy chainimmunoglobulin constant region genes. By “constant light chain” or“light chain constant region” as used herein is meant the region of anantibody encoded by the kappa (Ckappa) or lambda (Clambda) light chains.The constant light chain typically comprises a single domain, and asdefined herein refers to positions 108-214 of Ckappa, or Clambda,wherein numbering is according to the EU index (Kabat et al., 1991,Sequences of Proteins of Immunological Interest, 5th Ed., United StatesPublic Health Service, National Institutes of Health, Bethesda). By“constant heavy chain” or “heavy chain constant region” as used hereinis meant the region of an antibody encoded by the mu, delta, gamma,alpha, or epsilon genes to define the antibody's isotype as IgM, IgD,IgG, IgA, or IgE, respectively. For full length IgG antibodies, theconstant heavy chain, as defined herein, refers to the N-terminus of theCH1 domain to the C-terminus of the CH3 domain, thus comprisingpositions 118-447, wherein numbering is according to the EU index.

By “Fab” or “Fab region” as used herein is meant the polypeptide thatcomprises the VH, CH1, VL, and CL immunoglobulin domains. Fab may referto this region in isolation, or this region in the context of apolypeptide, multispecific polypeptide or ABD, or any other embodimentsas outlined herein.

By “single-chain Fv” or “scFv” as used herein are meant antibodyfragments comprising the VH and VL domains of an antibody, wherein thesedomains are present in a single polypeptide chain. Generally, the Fvpolypeptide further comprises a polypeptide linker between the VH and VLdomains which enables the scFv to form the desired structure for antigenbinding. Methods for producing scFvs are well known in the art. For areview of methods for producing scFvs see Pluckthun in The Pharmacologyof Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.Springer-Verlag, New York, pp. 269-315 (1994).

By “Fv” or “Fv fragment” or “Fv region” as used herein is meant apolypeptide that comprises the VL and VH domains of a single antibody.

By “Fc” or “Fc region”, as used herein is meant the polypeptidecomprising the constant region of an antibody excluding the firstconstant region immunoglobulin domain. Thus Fc refers to the last twoconstant region immunoglobulin domains of IgA, IgD, and IgG, and thelast three constant region immunoglobulin domains of IgE and IgM, andthe flexible hinge N-terminal to these domains. For IgA and IgM, Fc mayinclude the J chain. For IgG, Fc comprises immunoglobulin domains Cy2(CH2) and Cy3 (CH3) and the hinge between Cy1 and Cy2. Although theboundaries of the Fc region may vary, the human IgG heavy chain Fcregion is usually defined to comprise residues C226, P230 or A231 to itscarboxyl-terminus, wherein the numbering is according to the EU index.Fc may refer to this region in isolation, or this region in the contextof an Fc polypeptide, as described below. By “Fc polypeptide” or“Fc-derived polypeptide” as used herein is meant a polypeptide thatcomprises all or part of an Fc region. Fc polypeptides include but isnot limited to antibodies, Fc fusions and Fc fragments.

By “variable region” as used herein is meant the region of an antibodythat comprises one or more Ig domains substantially encoded by any ofthe VL (including Vkappa (VK) and Vlambda) and/or VH genes that make upthe light chain (including kappa and lambda) and heavy chainimmunoglobulin genetic loci respectively. A light or heavy chainvariable region (VL or VH) consists of a “framework” or “FR” regioninterrupted by three hypervariable regions referred to as“complementarity determining regions” or “CDRs”. The extent of theframework region and CDRs have been precisely defined, for example as inKabat (see

“Sequences of Proteins of Immunological Interest,” E. Kabat et al., U.S.Department of Health and Human Services, (1983)), and as in Chothia. Theframework regions of an antibody, that is the combined framework regionsof the constituent light and heavy chains, serves to position and alignthe CDRs, which are primarily responsible for binding to an antigen.

The term “specifically binds to” means that an antibody or polypeptidecan bind preferably in a competitive binding assay to the bindingpartner, e.g. NKp46, as assessed using either recombinant forms of theproteins, epitopes therein, or native proteins present on the surface ofisolated target cells. Competitive binding assays and other methods fordetermining specific binding are further described below and are wellknown in the art.

When an antibody or polypeptide is said to “compete with” a particularmonoclonal antibody (e.g. NKp46-1, -2, -4, -6 or -9 in the context of ananti-NKp46 mono- or bi-specific antibody), it means that the antibody orpolypeptide competes with the monoclonal antibody in a binding assayusing either recombinant target (e.g. NKp46) molecules or surfaceexpressed target (e.g. NKp46) molecules. For example, if a test antibodyreduces the binding of NKp46-1, -2, -4, -6 or -9 to a NKp46 polypeptideor NKp46-expressing cell in a binding assay, the antibody is said to“compete” respectively with NKp46-1, -2, -4, -6 or -9.

The term “affinity”, as used herein, means the strength of the bindingof an antibody or polypeptide to an epitope. The affinity of an antibodyis given by the dissociation constant K_(D), defined as[Ab]×[Ag]/[Ab-Ag], where [Ab-Ag] is the molar concentration of theantibody-antigen complex, [Ab] is the molar concentration of the unboundantibody and [Ag] is the molar concentration of the unbound antigen. Theaffinity constant K_(A) is defined by 1/K_(D). Preferred methods fordetermining the affinity of mAbs can be found in Harlow, et al.,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1988), Coligan et al., eds., Current Protocolsin Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y.,(1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), whichreferences are entirely incorporated herein by reference. One preferredand standard method well known in the art for determining the affinityof mAbs is the use of surface plasmon resonance (SPR) screening (such asby analysis with a BIAcore™ SPR analytical device).

Within the context of this invention a “determinant” designates a siteof interaction or binding on a polypeptide.

The term “epitope” refers to an antigenic determinant, and is the areaor region on an antigen to which an antibody or polypeptide binds. Aprotein epitope may comprise amino acid residues directly involved inthe binding as well as amino acid residues which are effectively blockedby the specific antigen binding antibody or peptide, i.e., amino acidresidues within the “footprint” of the antibody. It is the simplest formor smallest structural area on a complex antigen molecule that cancombine with e.g., an antibody or a receptor. Epitopes can be linear orconformational/structural. The term “linear epitope” is defined as anepitope composed of amino acid residues that are contiguous on thelinear sequence of amino acids (primary structure). The term“conformational or structural epitope” is defined as an epitope composedof amino acid residues that are not all contiguous and thus representseparated parts of the linear sequence of amino acids that are broughtinto proximity to one another by folding of the molecule (secondary,tertiary and/or quaternary structures). A conformational epitope isdependent on the 3-dimensional structure. The term ‘conformational’ istherefore often used interchangeably with ‘structural’.

By “amino acid modification” herein is meant an amino acid substitution,insertion, and/or deletion in a polypeptide sequence. An example ofamino acid modification herein is a substitution. By “amino acidmodification” herein is meant an amino acid substitution, insertion,and/or deletion in a polypeptide sequence. By “amino acid substitution”or “substitution” herein is meant the replacement of an amino acid at agiven position in a protein sequence with another amino acid. Forexample, the substitution Y50W refers to a variant of a parentpolypeptide, in which the tyrosine at position 50 is replaced withtryptophan. A “variant” of a polypeptide refers to a polypeptide havingan amino acid sequence that is substantially identical to a referencepolypeptide, typically a native or “parent” polypeptide. The polypeptidevariant may possess one or more amino acid substitutions, deletions,and/or insertions at certain positions within the native amino acidsequence.

“Conservative” amino acid substitutions are those in which an amino acidresidue is replaced with an amino acid residue having a side chain withsimilar physicochemical properties. Families of amino acid residueshaving similar side chains are known in the art, and include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

The term “identity” or “identical”, when used in a relationship betweenthe sequences of two or more polypeptides, refers to the degree ofsequence relatedness between polypeptides, as determined by the numberof matches between strings of two or more amino acid residues.“Identity” measures the percent of identical matches between the smallerof two or more sequences with gap alignments (if any) addressed by aparticular mathematical model or computer program (i.e., “algorithms”).Identity of related polypeptides can be readily calculated by knownmethods. Such methods include, but are not limited to, those describedin Computational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M.and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carilloet al., SIAM J. Applied Math. 48, 1073 (1988).

Preferred methods for determining identity are designed to give thelargest match between the sequences tested. Methods of determiningidentity are described in publicly available computer programs.Preferred computer program methods for determining identity between twosequences include the GCG program package, including GAP (Devereux etal., Nucl. Acid. Res. 12, 387 (1984); Genetics Computer Group,University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA(Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTX programis publicly available from the National Center for BiotechnologyInformation (NCBI) and other sources (BLAST Manual, Altschul et al.NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra). The well knownSmith Waterman algorithm may also be used to determine identity.

An “isolated” molecule is a molecule that is the predominant species inthe composition wherein it is found with respect to the class ofmolecules to which it belongs (i.e., it makes up at least about 50% ofthe type of molecule in the composition and typically will make up atleast about 70%, at least about 80%, at least about 85%, at least about90%, at least about 95%, or more of the species of molecule, e.g.,peptide, in the composition). Commonly, a composition of a polypeptidewill exhibit 98%, 98%, or 99% homogeneity for polypeptides in thecontext of all present peptide species in the composition or at leastwith respect to substantially active peptide species in the context ofproposed use.

In the context herein, “treatment” or “treating” refers to preventing,alleviating, managing, curing or reducing one or more symptoms orclinically relevant manifestations of a disease or disorder, unlesscontradicted by context. For example, “treatment” of a patient in whomno symptoms or clinically relevant manifestations of a disease ordisorder have been identified is preventive or prophylactic therapy,whereas “treatment” of a patient in whom symptoms or clinically relevantmanifestations of a disease or disorder have been identified generallydoes not constitute preventive or prophylactic therapy.

As used herein, “NK cells” refers to a sub-population of lymphocytesthat is involved in non-conventional immunity. NK cells can beidentified by virtue of certain characteristics and biologicalproperties, such as the expression of specific surface antigensincluding CD56 and/or NKp46 for human NK cells, the absence of thealpha/beta or gamma/delta TCR complex on the cell surface, the abilityto bind to and kill cells that fail to express “self” MHC/HLA antigensby the activation of specific cytolytic machinery, the ability to killtumor cells or other diseased cells that express a ligand for NKactivating receptors, and the ability to release protein moleculescalled cytokines that stimulate or inhibit the immune response. Any ofthese characteristics and activities can be used to identify NK cells,using methods well known in the art. Any subpopulation of NK cells willalso be encompassed by the term NK cells. Within the context herein“active” NK cells designate biologically active NK cells, including NKcells having the capacity of lysing target cells or enhancing the immunefunction of other cells. NK cells can be obtained by various techniquesknown in the art, such as isolation from blood samples, cytapheresis,tissue or cell collections, etc. Useful protocols for assays involvingNK cells can be found in Natural Killer Cells Protocols (edited byCampbell K S and Colonna M). Human Press. pp. 219-238 (2000).

As used herein, an agent that has “agonist” activity at Nkp46 is anagent that can cause or increase “NKp46 signaling”. “Nkp46 signaling”refers to an ability of a NKp46 polypeptide to activate or transduce anintracellular signaling pathway. Changes in NKp46 signaling activity canbe measured, for example, by assays designed to measure changes in NKp46signaling pathways, e.g. by monitoring phosphorylation of signaltransduction components, assays to measure the association of certainsignal transduction components with other proteins or intracellularstructures, or in the biochemical activity of components such askinases, or assays designed to measure expression of reporter genesunder control of NKp46-sensitive promoters and enhancers, or indirectlyby a downstream effect mediated by the NKp46 polypeptide (e.g.activation of specific cytolytic machinery in NK cells). Reporter genescan be naturally occurring genes (e.g. monitoring cytokine production)or they can be genes artificially introduced into a cell. Other genescan be placed under the control of such regulatory elements and thusserve to report the level of NKp46 signaling.

“NKp46” refers to a protein or polypeptide encoded by the Ncr1 gene orby a cDNA prepared from such a gene. Any naturally occurring isoform,allele or variant is encompassed by the term NKp46 polypeptide (e.g., anNKp46 polypeptide 90%, 95%, 98% or 99% identical to SEQ ID NO 1, or acontiguous sequence of at least 20, 30, 50, 100 or 200 amino acidresidues thereof). The 304 amino acid residue sequence of human NKp46(isoform a) is shown as follows:

(SEQ ID NO: 1) MSSTLPALLC VGLCLSQRIS AQQQTLPKPF IWAEPHFMVPKEKQVTICCQ GNYGAVEYQL HFEGSLFAVD RPKPPERINKVKFYIPDMNS RMAGQYSCIY RVGELWSEPS NLLDLVVTEMYDTPTLSVHP GPEVISGEKV TFYCRLDTAT SMFLLLKEGRSSHVQRGYGK VQAEFPLGPV TTAHRGTYRC FGSYNNHAWSFPSEPVKLLV TGDIENTSLA PEDPTFPADT WGTYLLTTETGLQKDHALWD HTAQNLLRMG LAFLVLVALV WFLVEDWLSR KRTRERASRA STWEGRRRLN TQTL.

SEQ ID NO: 1 corresponds to NCBI accession number NP_004820, thedisclosure of which is incorporated herein by reference. The human NKp46mRNA sequence is described in NCBI accession number NM_004829, thedisclosure of which is incorporated herein by reference.

Producing Polypeptides

The antigen binding domains used in the proteins described herein can bereadily derived a variety of immunoglobulin or non-immunoglobulinscaffolds, for example affibodies based on the Z-domain ofstaphylococcal protein A, engineered Kunitz domains, monobodies oradnectins based on the 10th extracellular domain of human fibronectinIII, anticalins derived from lipocalins, DARPins (desiged ankyrin repeatdomains, multimerized LDLR-A module, avimers or cysteine-rich knottinpeptides. See, e.g., Gebauer and Skerra (2009) Current Opinion inChemical Biology 13:245-255, the disclosure of which is incorporatedherein by reference.

Variable domains are commonly derived from antibodies (immunoglobulinchains), for example in the form of associated VL and VH domains foundon two polypeptide chains, or single chain antigen binding domains suchas scFv, a V_(H) domain, a V_(L) domain, a dAb, a V-NAR domain or aV_(H)H domain. The an antigen binding domain (e.g,. ABD₁ and ABD₂) canalso be readily derived from antibodies as a Fab.

Typically, antibodies are initially obtained by immunization of anon-human animal, e.g., a mouse, with an immunogen comprising apolypeptide, or a fragment or derivative thereof, typically animmunogenic fragment, for which it is desired to obtain antibodies (e.g.a human polypeptide). The step of immunizing a non-human mammal with anantigen may be carried out in any manner well known in the art forstimulating the production of antibodies in a mouse (see, for example,E. Harlow and D. Lane, Antibodies: A Laboratory Manual., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1988), the entiredisclosure of which is herein incorporated by reference). Otherprotocols may also be used as long as they result in the production of Bcells expressing an antibody directed to the antigen used inimmunization. Lymphocytes from a non-immunized non-human mammal may alsobe isolated, grown in vitro, and then exposed to the immunogen in cellculture. The lymphocytes are then harvested and the fusion stepdescribed below is carried out. For exemplarymonoclonal antibodies, thenext step is the isolation of splenocytes from the immunized non-humanmammal and the subsequent fusion of those splenocytes with animmortalized cell in order to form an antibody-producing hybridoma. Thehybridoma colonies are then assayed for the production of antibodiesthat specifically bind to the polypeptide against which antibodies aredesired. The assay is typically a colorimetric ELISA-type assay,although any assay may be employed that can be adapted to the wells thatthe hybridomas are grown in. Other assays include radioimmunoassays orfluorescence activated cell sorting. The wells positive for the desiredantibody production are examined to determine if one or more distinctcolonies are present. If more than one colony is present, the cells maybe re-cloned and grown to ensure that only a single cell has given riseto the colony producing the desired antibody. After sufficient growth toproduce the desired monoclonal antibody, the growth media containingmonoclonal antibody (or the ascites fluid) is separated away from thecells and the monoclonal antibody present therein is purified.Purification is typically achieved by gel electrophoresis, dialysis,chromatography using protein A or protein G-Sepharose, or an anti-mouseIg linked to a solid support such as agarose or Sepharose beads (alldescribed, for example, in the Antibody Purification Handbook,Biosciences, publication No. 18-1037-46, Edition AC, the disclosure ofwhich is hereby incorporated by reference).

Human antibodies may also be produced by using, for immunization,transgenic animals that have been engineered to express a human antibodyrepertoire (Jakobovitz et Nature 362 (1993) 255), or by selection ofantibody repertoires using phage display methods. For example, aXenoMouse (Abgenix, Fremont, Calif.) can be used for immunization. AXenoMouse is a murine host that has had its immunoglobulin genesreplaced by functional human immunoglobulin genes. Thus, antibodiesproduced by this mouse or in hybridomas made from the B cells of thismouse, are already humanized. The XenoMouse is described in U.S. Pat.No. 6,162,963, which is herein incorporated in its entirety byreference.

Antibodies may also be produced by selection of combinatorial librariesof immunoglobulins, as disclosed for instance in (Ward et al. Nature,341 (1989) p. 544, the entire disclosure of which is herein incorporatedby reference). Phage display technology (McCafferty et al (1990) Nature348:552-553) can be used to produce antibodies from immunoglobulinvariable (V) domain gene repertoires from unimmunized donors. See, e.g.,Griffith et al (1993) EMBO J. 12:725-734; U.S. Pat. Nos. 5,565,332;5,573,905; 5,567,610; 5,229,275). When combinatorial libraries comprisevariable (V) domain gene repertoires of human origin, selection fromcombinatorial libraries will yield human antibodies.

Additionally, a wide range of antibodies are available in the scientificand patent literature, including DNA and/or amino acid sequences, orfrom commercial suppliers. Antibodies will typically be directed to apre-determined antigen. Examples of antibodies include antibodies thatrecognize an antigen expressed by a target cell that is to beeliminated, for example a proliferating cell or a cell contributing to apathology. Examples include antibodies that recognize tumor antigens,microbial (e.g. bacterial) antigens or viral antigens.

Antigen binding domains that bind NKp46 can be derived from theanti-NKp46 antibodies provided herein (see section “CDR Sequences”).Variable regions can be used directly, or can be modified by selectinghypervariable or CDR regions from the NKp46 antibodies and placing theminto an appropriate VL or VH framework, for example human frameworks.Antigen binding domains that bind NKp46 can also be derived de novousing methods for generating antibodies. Antibodies can be tested forbinding to NKp46 polypeptides. In one aspect of any embodiment herein, apolypeptide (e.g. multispecific polypeptide, bispecific or monospecificantibody) that binds to NKp46 will be capable of binding NKp46 expressedon the surface of a cell, e.g. native NKp46 expressed by a NK cell.

Antigen binding domains (ABDs) that bind antigens of interest can beselected based on the desired cellular target, and may include forexample cancer antigens, bacterial or viral antigens, etc. As usedherein, the term “bacterial antigen” includes, but is not limited to,intact, attenuated or killed bacteria, any structural or functionalbacterial protein or carbohydrate, or any peptide portion of a bacterialprotein of sufficient length (typically about 8 amino acids or longer)to be antigenic. Examples include gram-positive bacterial antigens andgram-negative bacterial antigens. In some embodiments the bacterialantigen is derived from a bacterium selected from the group consistingof Helicobacter species, in particular Helicobacter pyloris; Boreliaspecies, in particular Borelia burgdorferi; Legionella species, inparticular Legionella pneumophilia; Mycobacteria s species, inparticular M. tuberculosis, M. avium, M. intracellulare, M. kansasii, M.gordonae; Staphylococcus species, in particular Staphylococcus aureus;Neisseria species, in particular N. gonorrhoeae, N. meningitidis;Listeria species, in particular Listeria monocytogenes; Streptococcusspecies, in particular S. pyogenes, S. agalactiae; S. faecalis; S.bovis, S. pneumonias; anaerobic Streptococcus species; pathogenicCampylobacter species; Enterococcus species; Haemophilus species, inparticular Haemophilus influenzue; Bacillus species, in particularBacillus anthracis; Corynebacterium species, in particularCorynebacterium diphtheriae; Erysipelothrix species, in particularErysipelothrix rhusiopathiae; Clostridium species, in particular C.perfringens, C. tetani; Enterobacter species, in particular Enterobacteraerogenes, Klebsiella species, in particular Klebsiella 1S. pneumoniae,Pasteurella species, in particular Pasteurella multocida, Bacteroidesspecies; Fusobacterium species, in particular Fusobacterium nucleatum;Streptobacillus species, in particular Streptobacillus moniliformis;Treponema species, in particular Treponema pertenue; Leptospira;pathogenic Escherichia species; and Actinomyces species, in particularActinomyces israelli.

As used herein, the term “viral antigen” includes, but is not limitedto, intact, attenuated or killed whole virus, any structural orfunctional viral protein, or any peptide portion of a viral protein ofsufficient length (typically about 8 amino acids or longer) to beantigenic. Sources of a viral antigen include, but are not limited toviruses from the families: Retroviridae (e.g., human immunodeficiencyviruses, such as HIV-1 (also referred to as HTLV-III, LAV orHTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP;Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses,human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g.,strains that cause gastroenteritis); Togaviridae (e.g., equineencephalitis viruses, rubella viruses); Flaviviridae (e.g., dengueviruses, encephalitis viruses, yellow fever viruses); Coronaviridae(e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitisviruses, rabies viruses); Filoviridae (e.g., ebola viruses);Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measlesvirus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenzaviruses); Bunyaviridae (e.g., Hantaan viruses, bunya viruses,phleboviruses and Nairo viruses); Arenaviridae (hemorrhagic feverviruses); Reoviridae (e.g., reoviruses, orbiviruses and rotaviruses);Bornaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae(parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses);Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus(HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpesvirus; Poxyiridae (variola viruses, vaccinia viruses, pox viruses); andIridoviridae (e.g., African swine fever virus); and unclassified viruses(e.g., the agent of delta hepatitis (thought to be a defective satelliteof hepatitis B virus), Hepatitis C; Norwalk and related viruses, andastroviruses). Alternatively, a viral antigen may be producedrecombinantly.

As used herein, the terms “cancer antigen” and “tumor antigen” are usedinterchangeably and refer to antigens that are differentially expressedby cancer cells and can thereby be exploited in order to target cancercells. Cancer antigens are antigens which can potentially stimulateapparently tumor-specific immune responses. Some of these antigens areencoded, although not necessarily expressed, by normal cells. Theseantigens can be characterized as those which are normally silent (i.e.,not expressed) in normal cells, those that are expressed only at certainstages of differentiation and those that are temporally expressed suchas embryonic and fetal antigens. Other cancer antigens are encoded bymutant cellular genes, such as oncogenes (e.g., activated ras oncogene),suppressor genes (e.g., mutant p53), fusion proteins resulting frominternal deletions or chromosomal translocations. Still other cancerantigens can be encoded by viral genes such as those carried on RNA andDNA tumor viruses.

The cancer antigens are usually normal cell surface antigens which areeither overexpressed or expressed at abnormal times. Ideally the targetantigen is expressed only on proliferative cells (e.g., tumor cells),however this is rarely observed in practice. As a result, targetantigens are usually selected on the basis of differential expressionbetween proliferative and healthy tissue. Antibodies have been raised totarget specific tumor related antigens including: Receptor TyrosineKinase-like Orphan Receptor 1 (ROR1), Cripto, CD4, CD20, CD30, CD19,CD33, CD38, CD47, Glycoprotein NMB, CanAg, Her2 (ErbB2/Neu), CD22(Siglec2), CD33 (Siglec3), CD79, CD138, CD171, PSCA, L1-CAM, PSMA(prostate specific membrane antigen), BCMA, CD52, CD56, CD80, CD70,E-selectin, EphB2, Melanotransferin, Mud 6 and TMEFF2. Examples ofcancer antigens also include B7-H3, B7-H4, B7-H6, PD-L1, MAGE,MART-1/Melan-A, gp100, major histocompatibility complex class I-relatedchain A and B polypeptides (MICA and MICB), adenosine deaminase-bindingprotein (ADAbp), cyclophilin b, colorectal associated antigen(CRC)-C017-1A/GA733, Killer-Ig Like Receptor 3DL2 (KIR3DL2), proteintyrosine kinase 7 (PTK7), receptor protein tyrosine kinase 3 (TYRO-3),nectins (e.g. nectin-4), major histocompatibility complex classI-related chain A and B polypeptides (MICA and MICB), proteins of theUL16-binding protein (ULBP) family, proteins of the retinoic acid earlytranscript-1 (RAET1) family, carcinoembryonic antigen (CEA) and itsimmunogenic epitopes CAP-1 and CAP-2, etv6, aml1, prostate specificantigen (PSA), T-cell receptor/CD3-zeta chain, MAGE-family of tumorantigens, GAGE-family of tumor antigens, anti-Mullerian hormone Type IIreceptor, delta-like ligand 4 (DLL4), DR5, ROR1 (also known as ReceptorTyrosine Kinase-Like Orphan Receptor 1 or NTRKR1 (EC 2.7.10.1), BAGE,RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, MUC family, VEGF, VEGF receptors,Angiopoietin-2, PDGF, TGF-alpha, EGF, EGF receptor, a member of thehuman EGF-like receptor family such as HER-2/neu, HER-3, HER-4 or aheterodimeric receptor comprised of at least one HER subunit, gastrinreleasing peptide receptor antigen, Muc-1, CA125, αvβ3 integrins, α5β1integrins, αIIbβ3-integrins, PDGF beta receptor, SVE-cadherin, IL-8,hCG, IL-6, IL-6 receptor, IL-15, α-fetoprotein, β-cadherin, α-catenin,β-catenin and γ-catenin, p120ctn, PRAME, NY-ESO-1, cdc27, adenomatouspolyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, p15,gp75, GM2 and GD2 gangliosides, viral products such as humanpapillomavirus proteins, imp-1, P1A, EBV-encoded nuclear antigen(EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40),SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, and c-erbB-2, although this is notintended to be exhaustive. In one aspect, the antigen of interest is aCD19 polypeptide; in one aspect, the multispecific protein comprises anscFv that binds CD19 comprising an amino acid sequence which is at least60%, 70%, 80%, 85%, 90% or 95% identical to the sequence of theanti-CD19 scFv of the Examples herein, or that comprises the heavy andlight chain CDR1, -2 and -3 of the anti-CD19 heavy and light chainvariable regions shown herein.

In one embodiment, the ABD binds to a cancer antigen, a viral antigen, amicrobial antigen, or an antigen present on an infected cell (e.g.virally infected) or on a pro-inflammatory immune cell. In oneembodiment, said antigen is a polypeptide selectively expressed oroverexpressed on a tumor cell, and infected cell or a pro-inflammatorycell. In one embodiment, said antigen is a polypeptide that wheninhibited, decreases the proliferation and/or survival of a tumor cell,an infected cell or a pro-inflammatory cell. For example, a first and/orsecond antibody or fragment can respectively bind anti-Her1 andanti-Her2. Anti-Her2 can be for example an antibody comprising the CDRsderived from Herceptin® (trastuzumab) or 2C4 (pertuzumab). Anti-Her2 andanti-Her1 (antibodies D1-5 and C3-101) amino acid sequences are shown inWO2011/069104.

The ABD which are incorporated into the polypeptides can be tested forany desired activity prior to inclusion in a multispecific NKp46-bindingprotein, for example the ABD can be tested for binding to an antigen ofinterest.

An ABD derived from an antibody will generally comprise at minimum ahypervariable region sufficient to confer binding activity. It will beappreciated that an ABD may comprise other amino acids or functionaldomains as may be desired, including but not limited to linker elements(e.g. linker peptides, CH1, CK or CA domains, hinges, or fragmentsthereof). In one example an ABD comprises an scFv, a V_(H) domain and aV_(L) domain, or a single domain antibody (nanobody or dAb) such as aV-NAR domain or a V_(H)H domain. Exemplary antibody formats are furtherdescribed herein and an ABD can be selected based on the desired format.

In any embodiment, an antigen binding domain can be obtained from ahumanized antibody in which residues from a complementary-determiningregion (CDR) of a human antibody are replaced by residues from a CDR ofthe original antibody (the parent or donor antibody, e.g. a murine orrat antibody) while maintaining the desired specificity, affinity, andcapacity of the original antibody. The CDRs of the parent antibody, someor all of which are encoded by nucleic acids originating in a non-humanorganism, are grafted in whole or in part into the beta-sheet frameworkof a human antibody variable region to create an antibody, thespecificity of which is determined by the engrafted CDRs. The creationof such antibodies is described in, e.g., WO 92/11018, Jones, 1986,Nature 321:522-525, Verhoeyen et al., 1988, Science 239:1534-1536. Anantigen binding domain can thus have non-human hypervariable regions orCDRs and human frameworks region sequences (optionally withbackmutations).

Once appropriate antigen binding domains having desired specificityand/or activity are identified, DNA encoding each of the or ABD can beseparately placed, in suitable arrangements, in an appropriateexpression vector, together with DNA encoding any elements such as anenzymatic recognition tag, or CH2 and CH3 domains and any other optionalelements (e.g. DNA encoding a hinge region) for transfection into anappropriate host. ABDs will be arranged in an expression vector, or inseparate vectors as a function of which type of polypeptide is to beproduced, so as to produce the Fc-polypeptides having the desireddomains operably linked to one another. The host is then used for therecombinant production of the multispecific polypeptide.

For example, a polypeptide fusion product can be produced from a vectorin which the first of the two ABD is operably linked (e.g. directly, viaa heavy or light chain CH1, CK or CA constant region and/or hingeregion) to the N-terminus of a CH2 domain, and the CH2 domain isoperably linked at its C-terminus to the N-terminus a CH3 domain. Thesecond of the two ABD can be linked to the polypeptide at eitherterminus, or can be on a second polypeptide chain that forms a dimer,e.g. heterodimer, with the polypeptide comprising the first ABD. Thepolypeptide may comprise a full length Fc domain.

The multispecific polypeptide can then be produced in an appropriatehost cell or by any suitable synthetic process. A host cell chosen forexpression of the multispecific polypeptide is an important contributorto the final composition, including, without limitation, the variationin composition of the oligosaccharide moieties decorating the protein inthe immunoglobulin CH2 domain. Thus, one aspect of the inventioninvolves the selection of appropriate host cells for use and/ordevelopment of a production cell expressing the desired therapeuticprotein such that the multispecific polypeptide retains at least partialFcRn binding but with decreased binding to a Fcγ receptor compared,e.g., to a wild type full length human IgG1 antibody. The host cell maybe of mammalian origin or may be selected from COS-1, COS-7, HEK293,BHK21, CHO, BSC-1, Hep G2, 653, SP2/0, 293, HeLa, myeloma, lymphoma,yeast, insect or plant cells, or any derivative, immortalized ortransformed cell thereof. Alternatively, the host cell may be selectedfrom a species or organism incapable of glycosylating polypeptides, e.g.a prokaryotic cell or organism, such as natural or engineered E. colispp., Klebsiella spp., or Pseudomonas spp.

Monomeric Proteins

Monomeric multispecific proteins can be produced according to a varietyof formats. In one example, a multispecific proteins comprises in asingle polypeptide chain a first antigen binding domain that binds toNKp46 and a second antigen binding domain that binds an antigen otherthan NKp46. In one embodiment, the antibody is a tandem scFv, optionallyfused to another polypeptide or amino acid sequence. In one embodiment,the single polypeptide chain further comprises an Fc domain (e.g. a fulllength Fc domain or a portion thereof), optionally wherein the Fc domainis interposed between the first and second antigen binding domains.

Monomeric bispecific Fc-derived polypeptides having advantageousproperties can be constructed that comprise: (a) an antigen bindingdomain that binds to NKp46; (b) an antigen binding domain that binds anantigen other than NKp46; and (c) at least a portion of a human Fcdomain, wherein the Fc domain (i) does not dimerize with anotherFc-derived polypeptide, (ii) is capable of binding to human FcRn and(iii) has decreased binding (or lacks binding) to a human Fcγ receptorcompared to a wild type human IgG1 Fc domain. Optionally, the Fc domainis interposed between the first and second ABD.

In one aspect of any embodiment, the first antigen binding domain and/orthe second antigen binding domain comprise a scFv, optionally where thescFv comprises human framework amino acid sequences. In one embodiment,provided is a monomeric bispecific Fc-derived polypeptide comprising:(a) a first scFv that binds to NKp46; (b) a second scFv that binds anantigen other than NKp46; and (c) at least a portion of a human Fcdomain, wherein the Fc domain (i) does not dimerize with anotherFc-derived polypeptide, (ii) is capable of binding to human FcRn and(iii) has decreased binding to a human Fcγ receptor compared to a wildtype human IgG1 Fc domain. Optionally, the Fc domain is interposedbetween the first and second scFv.

When the polypeptide fusion product comprising the two ABDs and at leasta portion of an Fc domain is a monomer, the CH3 domains may be arrangedand/or comprise amino acid modification to prevent CH3-CH3 dimerization.In one embodiment, the CH3 domain comprises mutations in the dimerinterface to prevent interchain CH3-CH3 dimerization. In anotherembodiment, the CH3 domain is a tandem CH3 domain (or the Fc domaincomprises a tandem CH3 domain) to prevent interchain CH3-CH3dimerization. Such monomers will retain partial FcRn binding (compared,e.g., to a wild type full length human IgG1 antibody), yet havedecreased human Fcγ receptor binding. Optionally the monomericpolypeptide is capable of binding to human FcRn with intermediateaffinity, e.g. retains binding to FcRn but has decreased binding to ahuman FcRn receptor compared to a full-length wild type human IgG1antibody. The Fc moiety may further comprise one or more amino acidmodifications, e.g. in the CH2 domain, that further decreases orsubstantially abolishes binding to one or more Fcγ receptors.

Optionally in any of the embodiments, the Fc domain comprises a CH2domain and a CH3 domain comprising one or more amino acid modificationssuch that the Fc domain which does not dimerize with another Fc-derivedpolypeptide (e.g. does not dimerize via interactions with another CH3domain).

In some embodiments of the polypeptides, the ABD that binds NKp46 willbe operably linked to the ABD that binds an antigen other than NKp46(e.g. the two ABDs are fused via a linker), and one of the two ABD willin turn be fused to a CH2 domain which is in turn fused (e.g. fused atits C-terminus) to a CH3 domain (or a CH3 which is in turn fused a CH2domain). In some embodiments, the first ABD will be operably linked tothe second. ABD via a peptide linker such that a tandem antigen bindingdomain is formed that comprises both ABDs.

Examples of such polypeptides may comprise a domain arrangement of anyone of the following:

(ABD₁)-(ABD₂)-CH2-CH3

(ABD₂)-(ABD₁)-CH2-CH3

CH2-CH3-(ABD₁)-(ABD₂)

CH2-CH3-(ABD₂)-(ABD₁)

wherein one of ABD₁ and ABD₁ binds an antigen of interest and the otherbinds NKp46, optionally wherein a CH1 domain or fragment thereof and/orhinge domain is placed between an ABD₁ and CH2 or between an ABD₂ andCH2. Optionally, each ABD comprises a VL and a VH domain. Optionally,any of the polypeptides comprises a tandem CH3 domain wherein a secondCH3 domain fused via a flexible linker to the C-terminal of the firstCH3 domain.

Optionally the ABDs are each scFv such that tandem scFv-containingpolypeptides are produced. The first and second ABDs can be linkedtogether by a linker of sufficient length to enable the ABDs to fold insuch a way as to permit binding to the respective antigen for which theABD is intended to bind. Suitable peptide linkers for use in linkingABD₁ to ABD₂, or for use in linking an ABD to a CH2 or CH3 are known inthe art, see, e.g. WO2007/073499, the disclosure of which isincorporated herein by reference. Examples of linker sequences include(G₄S)-wherein x is an integer (e.g. 1, 2, 3, 4, or more). The tandemantigen binding domain can thus for example have the structure(ABD₁-peptide linker-ABD₂-peptide linker-(monomeric CH2-CH3domain-containing polypeptide)). For example, the polypeptide maycomprise, as a fusion product, the structure (scFv₁-peptidelinker-scFv₂-peptide linker-CH2-CH3), wherein each element is fused tothe following element.

In any domain arrangement presented herein, the ABD that binds NKp46 maybe represented by either ABD₁ or ABD₂, and the ABD that binds an antigenof interest may be represented by either ABD₁ or ABD₂, so long as one ofthe ABD₁ or ABD₂ binds NKp46 and the other binds antigen of interest.

In some embodiments of the polypeptides having a first antigen bindingdomain (ABD₁) and second antigen binding domain (ABD₂), one of the twoABD will in turn be fused, optionally via intervening amino acids, toone end of an Fc domain (e.g. comprising a full or partial CH2 and afull or partial CH3 domain) and the other of the two ABD is fused,optionally via intervening amino acids, to opposite end of the Fcdomain. In some embodiments, an ABD will be linked to the CH2 domain viaa linker (e.g. comprising a full or partial hinge region and/or a fullor partial CH1 domain). Such polypeptides will have the advantage, interalia, that antibody VL and VH domains that are not functional whenconstructed as a tandem scFv but are functional in single scFv form canbe readily used. The polypeptides may comprise a domain arrangement ofany one of the following:

(ABD₁)-CH2-CH3-(ABD₂)

(ABD₂)-CH2-CH3-(ABD₁)

wherein one of ABD₁ and ABD₁ binds an antigen of interest and the otherbinds NKp46, optionally wherein a CH1 domain and/or hinge domain isplaced between an ABD₁ and CH2 or between an ABD₂ and CH2. Optionally,each ABD comprises a VL and a VH domain. Optionally, any of thepolypeptides has a second CH3 domain fused via a flexible linker to theC-terminal of the first CH3 domain. Examples of such polypeptides areshown as formats 1, 3 and 4 in FIG. 6A.

The monomeric Fc-derived polypeptides that have at least a portion of ahuman Fc domain can advantageously comprise a CH2 domain that does notsubstantially bind to an FcγIIIA polypeptide (CD16) and a CH3 domain,wherein said CH3 domain comprises a modified CH3 dimer interface (e.g. amutations in the CH3 dimer interface) to prevent dimerization withanother Fc-derived polypeptide.

In one embodiment of any of the polypeptides or methods herein, the CH3domain comprises an amino acid substitution at 1, 2, 3, 4, 5, 6 or 7 ofthe positions L351, T366, L368, P395, F405, T407 (or Y407) and/or K409(EU numbering as in Kabat).

Another configuration for a CH3 domain that can be used in a monomericmultispecific protein is a tandem CH3 domain (see e.g. format 3 and 4 inFIG. 6A). A tandem CH3 domain comprises a first and a second CH3 domain,wherein the two CH3 domains associate with one another via non-covalentinteractions. In one embodiment, the two CH3 domains associate with oneanother via the CH3 dimerization interface of each CH3 domain. In oneembodiment, the polypeptide chain does not dimerize with anotherpolypeptide chain comprising an Fc domain. An Fc domain that comprise atandem CH3 domain will interact with neonatal Fc receptor (FcRn) butwill have low or no binding to human Fcγ receptors, notably CD16.

In one embodiment of any aspect herein, a first CH3 domain is connectedto a second CH3 domain by a linker. The tandem CH3 domains can thus beplaced on the same polypeptide chain so as to have the domainarrangement, from N-terminus to C-terminus, as follows:

—CH3-linker-CH3-.

The linker will be a flexible linker (e.g. peptide linker). In oneembodiment the linker permits the CH3 domains to associate with oneanother by non-covalent interactions. In one embodiment, the linker is apeptide linker having 10-50 amino acid residues. In one embodiment, thelinker has the formula (G₄S)_(x). Optionally, x is 2, 3, 4, 5 or 6. Inany of the embodiments, each CH3 domain is independently a full-lengthand/or native CH3 domain, or a fragment or modified CH3 domain whichretains a functional CH3 dimerization interface.

Examples of domain arrangements of monomeric proteins of the inventiontherefore include any one of the following:

(ABD₁)-CH2-CH3-linker-CH3-(ABD₂)

(ABD₂)-CH2-CH3-linker-CH3-(ABD₁)

(ABD₁)-(ABD₂)-CH2-CH3-linker-CH3

(ABD₂)-(ABD₁)-CH2-CH3-linker-CH3

CH2-CH3-linker-CH3-(ABD₁)-(ABD₂)

CH2-CH3-linker-CH3-(ABD₂)-(ABD₁)

Multimeric Proteins

Multimeric bispecific proteins such as heterodimers, heterotrimers andtetramers (the latter including for example antibodies with two heavychains and two light chains) can be produced according to a variety offormats.

In one advantageous format for NKp46 antibodies, the multimericpolypeptide is capable of binding to human FcRn and has decreasedbinding to a human Fcγ receptor (e.g. CD16, CD32 and/or CD64) compared,e.g., to a full length wild type human IgG1 antibody. When thepolypeptide comprising the two ABDs is a multimer, Fc moieties with atleast partial FcRn binding and decreased or abolished human Fcγ receptorbinding can be obtained through the use of suitable CH2 and/or CH3domains, as further described herein. In one embodiment, an Fc moiety isderived from a human IgG4 isotype constant region, as IgG4 based Fcdomains will retain substantial FcRn binding but have reduced Fcγreceptor binding. In one embodiment, an Fc moiety may be obtained byproduction of the polypeptide in a host cell or by a process that doesnot yield N297-linked glycosylation, e.g. a bacterial cell. In oneembodiment, an Fc moiety comprises one or more amino acid modifications,e.g. in the CH2 domain, that decreases binding to one or more Fcγreceptors and retains at least partial FcRn binding.

In one embodiment, exemplary heterodimer molecules can have a domainarrangement:

wherein V₁ and V₂ are single variable domains (e.g. V_(H) domain, aV_(L) domain, a dAb, a V-NAR domain or a V_(H)H domain), and one of V₁and V₂ binds NKp46 and the other binds an antigen of interest.Optionally, the CH3 domain is a tandem CH3 domain or a CH3 domainmodified to prevent CH3-CH3 dimerization.

In one embodiment, exemplary heterodimer molecules can have a domainarrangement:

wherein V₁ and V₂ are single variable domains (e.g. V_(H) domain, aV_(L) domain, a dAb, a V-NAR domain or a V_(H)H domain), and one of V₁and V₂ binds NKp46 and the other binds an antigen of interest.

In one embodiment, exemplary heterodimer molecules can have a domainarrangement:

wherein V_(1a), V_(1b), V_(2a) and V_(2b) are each a V_(H) domain or aV_(L) domain, and wherein one of V_(1a) and V_(1b) is a VH and the otheris a VL such that V_(1a) and V_(1b) form a first antigen binding domain(ABD), wherein one of V_(2a) and V_(2b) is a VH and the other is a VLsuch that V_(2a) and V_(2b) form a second ABD, wherein one of the ABDbinds NKp46 and the other binds an antigen of interest. Optionally theCH3 domain is a tandem CH3 domain or a CH3 domain modified to preventCH3-CH3 dimerization. Each pair of V domains can be separated by alinker peptide (e.g. to form an scFv).

In one embodiment, exemplary heterodimer molecules can have a domainarrangement:

wherein V_(a-1), V_(b-1), V_(a-2) and V_(b-2) are each a V_(H) domain ora V_(L) domain, and wherein one of V_(a-1) and V_(b-1) is a VH and theother is a VL such that V_(a-1) and V_(b-1) form a first antigen bindingdomain (ABD), wherein one of V_(a-2) and V_(b-2) is a VH and the otheris a VL such that V_(a-2) and V_(b-2) form a second antigen bindingdomain, wherein one of the ABD binds NKp46 and the other binds anantigen of interest. In one variant of the foregoing, any of, or each ofthe V_(a-1), V_(b-1), V_(a-2) and V_(b-2) are an scFv (made up of twovariable domains). Each pair of V domains can be separated by a linkerpeptide (e.g. to form an scFv).

In similar approaches, trimers can be constructed. Exemplaryheterotrimer molecules can have a domain arrangement:

wherein the first/central chain and the second chain associate byCH3-CH3 dimerization and first/central chain and the third chainassociate by the CH1 or CK dimerization, wherein the domains of thefirst/central chain and the third chain are selected to be complementaryto permit the first and third chains to associate by CH1-CKdimerization, and wherein V_(a-1), V_(b-1), V_(a-2) and V_(b-2) are eacha V_(H) domain or a V_(L) domain, and wherein one of V_(a-1) and V_(b-1)is a VH and the other is a VL such that V_(a-1) and V_(b-1) form a firstantigen binding domain (ABD), wherein one of V_(a-2) and V_(b-2) is a VHand the other is a VL such that V_(a-2) and V_(b-2) form a secondantigen binding domain (e.g. an scFv wherein V_(a-2) and V_(b-2) areseparated by a linker), wherein one of the ABD binds NKp46 and the otherbinds an antigen of interest. Optionally, CH3 domains comprise aminoacid substitutions, wherein the CH3 domain interface of the antibody Fcregion is mutated to create altered charge polarity across the Fc dimerinterface such that co-expression of electrostatically matched Fc chainssupport favorable attractive interactions thereby promoting desired Fcheterodimer formation, whereas unfavorable repulsive charge interactionssuppress unwanted Fc homodimer formation.

In other aspects, heterodimeric or heterotrimeric polypeptides with twoABDs separated by an interposed Fc domain can be produced in which oneor two chains each associate with a central chain by CH1-CKheterodimerization. Such multimers may be composed of a central (first)polypeptide chain comprising two immunoglobulin variable domains thatare part of separate antigen binding domains of different antigenspecificities, with an Fc domain interposed between the twoimmunoglobulin variable domains on the polypeptide chain, and a CH1 orCK constant domain placed on the polypeptide chain adjacent to avariable domain.

The first (central) polypeptide chain will provide one variable domainthat will, together with a complementary variable domain on a secondpolypeptide chain, form an antigen binding domain specific for one (e.g.a first) antigen of interest. The first (central) polypeptide chain willalso provide a second variable domain (placed on the opposite end of theinterposed Fc domain) that will be paired with a complementary variabledomain to form an antigen binding domain specific for another (e.g. asecond) antigen of interest; the variable domain that is complementaryto the second variable domain can be placed on the central polypeptide(e.g. adjacent to the second variable domain in a tandem variable domainconstruct such as an scFv), or can be placed on a separate polypeptidechain, notably a third polypeptide chain. The second (and third, ifpresent) polypeptide chains will associate with the central polypeptidechain by CH1-CK heterodimerization, forming interchain disulfide bondsbetween respective hinge domains and between complementary CH1 and CKdomains, with a primary multimeric polypeptide being formed so long asCH/CK and VH/VK domains are chosen to give rise to a preferreddimerization configuration that results preferentially in the desiredVH-VL pairings. Remaining unwanted pairings can remain minimal duringproduction and removed during purification steps. In a trimer, or whenpolypeptides are constructed for preparation of a trimer, there willgenerally be one polypeptide chain that comprises a non-naturallyoccurring VH—CK or VK—CH1 domain arrangement.

Examples of the domain arrangements (N- to C-terminal) of centralpolypeptide chains for use in such heterodimeric proteins include:

V_(a-1)-(CH1 or CK)_(a)—Fc domain-V_(a-2)-V_(b-2);

and

V_(a-2)-V_(b-2)-Fc domain-(CH1 or CK)_(a)

wherein V_(a-1) is a light chain or heavy chain variable domain, andwherein one of V_(a-2) and V_(b-2) is a light chain variable domain andthe other is a heavy chain variable domain.

Further examples include:

V_(a-1)-(CH1 or CK)_(a)—Fc domain-V_(b);

and

V_(b)-Fc domain-V_(a-1)-(CH1 or CK)_(a)

wherein V_(b) is a single variable domain (e.g. dAb, VhH).

The Fc domain of the central chain may be a full Fc domain (CH2-CH3) ora portion thereof sufficient to confer the desired functionality (e.g.FcRn binding). A second polypeptide chain will then be configured whichwill comprise an immunoglobulin variable domain and a CH1 or CK constantregion, e.g., a (CH1 or CK)_(b) unit, selected so as to permit CH1-CKheterodimerization with the central polypeptide chain; theimmunoglobulin variable domain will be selected so as to complement thevariable domain of the central chain that is adjacent to the CH1 or CKdomain, whereby the complementary variable domains form an antigenbinding domain for a first antigen of interest.

For example, a second polypeptide chain can comprise a domainarrangement:

V_(b-1)-(CH1 or CK)_(b),

or

V_(b-1)-(CH1 or CK)_(b)—Fc domain

such that the (CH1 or CK)₂ dimerizes with the (CH1 or CK)₁ on thecentral chain, and the V_(b-1) forms an antigen binding domain togetherwith V_(a-1) of the central chain. If V_(a-1) of the central chain is alight chain variable domain, V_(b-1) will be a heavy chain variabledomain; and if V_(a-1) of the central chain is a heavy chain variabledomain, V_(b-1) will be a light chain variable domain.

The antigen binding domain for the second antigen of interest can thenbe formed from V_(a-2) and V_(b-2) which are configured as tandemvariable domains on the central chain forming the antigen binding domainfor the second antigen of interest (e.g. a heavy chain variable domain(VH) and a light chain (kappa) variable domain (VK), for example formingan scFv unit). The antigen binding domain for the second antigen ofinterest can also alternatively be formed from a single variable domainV_(b) present on the central chain.

The resulting heterodimer can for example have the configuration asfollows (see also Examples of such proteins shown as formats 2, 11 and12 shown in FIGS. 6A and 6C):

wherein one of V_(a-1) of the first polypeptide chain and V_(b-1) of thesecond polypeptide chain is a light chain variable domain and the otheris a heavy chain variable domain, and wherein one of V_(a-2) and V_(b-2)is a light chain variable domain and the other is a heavy chain variabledomain.

The resulting heterodimer can in another example have the configurationas follows (see also Examples of such proteins shown as format 10 shownin FIG. 6B):

wherein one of V_(a-1) of the first polypeptide chain and V_(b-1) of thesecond polypeptide chain is a light chain variable domain and the otheris a heavy chain variable domain, and wherein one of V_(a-2) and V_(b-2)is a light chain variable domain and the other is a heavy chain variabledomain.

The resulting heterodimer can in another example have the configurationas follows (see also Examples of such proteins shown as formats 13 and14 shown in FIGS. 6D and 6E):

wherein one of V_(a-1) of the first polypeptide chain and V_(b-1) of thesecond polypeptide chain is a light chain variable domain and the otheris a heavy chain variable domain, and wherein one of V_(a-2) and V_(b-2)is a light chain variable domain and the other is a heavy chain variabledomain.

In one embodiment, the heterodimeric bispecific Fc-derived polypeptidecomprises a domain arrangement of one of the following, optionallywherein one or both hinge domains are replaced by a peptide linker,optionally wherein the Fc domain is fused to anti-NKp46 scFv via apeptide linker):

Examples of domain arrangement for the heterodimeric polypeptide formedinclude but are not limited to those in the table below:

Heterotrimeric proteins can for example be formed by using a central(first) polypeptide chain comprising a first variable domain (V) fusedto a first CH1 or CK constant region, a second variable domain (V) fusedto a second CH1 or CK constant region, and an Fc domain or portionthereof interposed between the first and second variable domains (i.e.the Fc domain is interposed between the first and second (V—(CH1/CK)units. For example, a central polypeptide chain for use in aheterotrimeric protein can have the domain arrangements (N- toC-terminal) as follows:

V_(a-1)-(CH1 or CK)_(a)—Fc domain-V_(a-2)-(CH1 or CK)_(b).

A second polypeptide chain can then comprise a domain arrangement (N- toC-terminal):

V_(b-1)-(CH1 or CK)_(c),

Or

V_(b-1)-(CH1 or CK)_(c)—Fc domain

such that the (CH1 or CK)_(c) dimerizes with the (CH1 or CK), on thecentral chain, and the V_(a-1) and V_(b-1) form an antigen bindingdomain.

A third polypeptide chain can then comprise a domain arrangement (N- toC-terminal):

V_(b-2)-(CH1 or CK)_(d),

such that the (CH1 or CK)_(d) dimerizes with the (CH1 or CK)_(b) unit onthe central chain, and the V_(a-2) and V_(b-2) form an antigen bindingdomain.

An example of a configuration of a resulting heterotrimer with a dimericFc domain (also shown as formats 5, 6, 7 and 16 in FIGS. 6D and 6E) hasa domain arrangement:

An example of a configuration of a resulting heterotrimer with amonomeric Fc domain (also shown as formats 8, 9 and 17 in FIGS. 6B and6C) has a domain arrangement:

Thus, in a configuration of a trimer polypeptide, the first polypeptidecan have two variable domains that each form an antigen binding domainwith a variable domain on a separate polypeptide chain (i.e. thevariable domain of the second and third chains), the second polypeptidechain has one variable domain, and the third polypeptide has onevariable domain.

A trimeric polypeptide may comprise:

(a) a first polypeptide chain comprising a first variable domain (V)fused to a first CH1 of CK constant region, a second variable domain (V)fused to a second CH1 of CK constant region, and an Fc domain or portionthereof interposed between the first and second variable domains;

(b) a second polypeptide chain comprising a variable domain fused at itsC-terminus to a CH1 or CK constant region selected to be complementaryto the first CH1 or CK constant region of the first polypeptide chainsuch that the first and second polypeptides form a CH1-CK heterodimer,and optionally an Fc domain; and

(c) a third polypeptide chain comprising a variable domain fused (e.g.at its C-terminus) to a CH1 or CK constant region, wherein the variabledomain and the constant region are selected to be complementary to thesecond variable domain and second CH1 or CK constant region of the firstpolypeptide chain such that the first and third polypeptides form aCH1-CK heterodimer bound by disulfide bond(s) formed between the CH1 orCK constant region of the third polypeptide and the second CH1 or CKconstant region of the first polypeptide, but not between the CH1 or CKconstant region of the third polypeptide and the first CH1 or CKconstant region of the first polypeptide wherein the first, second andthird polypeptides form a CH1-CK heterotrimer, and wherein the firstvariable domain of the first polypeptide chain and the variable domainof the second polypeptide chain form an antigen binding domain specificfor a first antigen of interest, and the second variable domain of thefirst polypeptide chain and the variable domain on the third polypeptidechain form an antigen binding domain specific for a second antigen ofinterest.

Examples of domain arrangement for the trimeric bispecific polypeptideformed from include but are not limited to:

In any of the domain arrangements, the Fc domain may comprise a CH2-CH3unit (a full length CH2 and CH3 domain or a fragment thereof). Inheterodimers or heterotrimers comprising two chains with Fc domains (adimeric Fc domain), the CH3 domain will be capable of CH3-CH3dimerization (e.g. a wild-type CH3 domain). In heterodimers orheterotrimers comprising only one chain with an Fc domain (monomeric Fcdomain), the Fc domain will be incapable of CH3-CH3 dimerization; forexample the CH3 domain(s) will have amino acid modification(s) in theCH3 dimer interface or the Fc domain will comprise a tandem CH3 domainincapable of CH3-CH3 dimerization. In one embodiment of any aspectherein, a first CH3 domain is connected to a second CH3 domain by alinker. The tandem CH3 domain may have the domain arrangement, fromN-terminus to C-terminus, as follows:

—CH3-linker-CH3-.

The linker in the tandem CH3 domain will be a flexible linker (e.g.peptide linker). In one embodiment the linker permits the CH3 domains toassociate with one another by non-covalent interactions. In oneembodiment, the linker is a peptide linker having 10-50 amino acidresidues. In one embodiment, the linker has the formula (G₄S)_(x).Optionally, x is 2, 3, 4, 5 or 6. In any of the embodiments, each CH3domain is independently a full-length and/or native CH3 domain, or afragment or modified CH3 domain which retains a functional CH3dimerization interface.

In some exemplary configurations, the multispecific protein can betetramers, e.g. tetramers with two heavy chains and two light chains. Insome embodiments, a “Fab-exchange” approach is used in which heavychains and attached light chains of different antibodies are swappedbetween two IgG4 or IgG4-like antibodies, see, e.g. WO2008/119353 andWO2011/131746, the disclosures of which are incorporated herein byreference. In some embodiments, a “knob-into-holes” approach is used inwhich the CH3 domain interface of the antibody Fc region is mutated sothat antibodies preferentially form heterodimers (further including theattached light chains). These mutations create altered charge polarityacross the Fc dimer interface such that co-expression ofelectrostatically matched Fc chains support favorable attractiveinteractions thereby promoting desired Fc heterodimer formation, whereasunfavorable repulsive charge interactions suppress unwanted Fc homodimerformation. See, e.g. WO2009/089004, the disclosure of which isincorporated herein by reference. When such hetero-multimeric antibodieshave Fc regions derived from a human IgG4 Fc region, the antibodies willretain substantial FcRn binding but have reduced Fcγ receptor binding.In one embodiment, the antibody lacks N-linked glycosylation at residueN297 (Kabat EU numbering)

In some embodiments, one of the ABDs is linked to (e.g. comprises avariable region linked to) a CH1 domain and the other of the ABDs islinked to (e.g. comprises a variable region linked to) a complementaryCK (or CA) constant domain, wherein the CH1 and CK (or CA) constantdomains associate to form a heterodimer molecule. For example, a firstand second ABD can advantageously be single variable domains (e.g. VhHdomains) having different antigen binding specificities (e.g., VhH₁ andVhH₂). VhH₁ can be fused to a CH1 domain and VhH₂ can be fused to a CKor CA domain. The V₁— CK (or CA) chain associates with a V₂—CH1 chainsuch that a Fab is formed. See, e.g., WO2006/064136 and WO2012/089814for examples of such antibodies without Fc domains, the disclosures ofwhich are incorporated herein by reference. The CH1 and/or CK domainscan then be linked to a CH2 domain, optionally via a hinge region (or alinker peptide, e.g., that has similar functional properties). The CH2domain(s) is/are then linked to a CH3 domain. The CH2-CH3 domains canthus optionally be embodied as a full-length Fc domain.

In some embodiments the protein is a tetrameric antibody comprising twolight chain and heavy chain pairs from different parental antibodies,comprising a modified CH3 domain interface so that antibodiespreferentially form heterodimers, optionally further wherein the Fcdomain is a human IgG4 Fc domain or a portion thereof, optionallycomprising one or more amino acid modifications

In one embodiment, tetrameric proteins are based two Fc containingchains (e.g. chains 1 and 2) to create a dimer via CH3-CH3 dimerizationand/or hinge dimerization, and two further chains (e.g. chains 3 and 4)each comprising a V—CH/CK unit that dimerizes with one of the twoFc-containing chains. For example such an exemplary tetramer moleculescan have a domain arrangement:

wherein V_(a-1), V_(b-1), V_(a-2) and V_(b-2) are each a V_(H) domain ora V_(L) domain, and wherein one of V_(a-1) and V_(b-1) is a VH and theother is a VL such that V_(a-1) and V_(b-1) form a first antigen bindingdomain (ABD), wherein one of V_(a-2) and V_(b-2) is a VH and the otheris a VL such that V_(a-2) and V_(b-2) form a second antigen bindingdomain. The CH1 and CK are selected such that chain 3 is capable ofassociating with chain 1 and chain 4 with chain 2.

For example such an exemplary tetramer molecules can have a domainarrangement:

wherein V_(a-1), V_(b-1), V_(a-2) and V_(b-2) are each a V_(H) domain ora V_(L) domain, and wherein one of V_(a-1) and V_(b-1) is a VH and theother is a VL such that V_(a-1) and V_(b-1) form a first antigen bindingdomain (ABD), wherein one of V_(a-2) and V_(b-2) is a VH and the otheris a VL such that V_(a-2) and V_(b-2) form a second antigen bindingdomain. The CH1 and CK are selected such that chain 3 is capable ofassociating with chain 1 and chain 4 with chain 2.

In any protein of the disclosure, a hinge region will typically bepresent on a polypeptide chain between a CH1 domain and a CH2 domain,and/or can be present between a CK domain and a CH2 domain. A hingeregion can optionally be replaced for example by a suitable linkerpeptide.

The proteins domains described in the present disclosure can optionallybe specified as being from N- to C-terminal. Protein arrangements of thedisclosure for purposes of illustration are shown from N-terminus (onthe left) to C-terminus. Domains can be referred to as fused to oneanother (e.g. a domain can be said to be fused to the C-terminus of thedomain on its left, and/or a domain can be said to be fused to theN-terminus of the domain on its right).

The proteins domains described in the present disclosure can be fused toone another directly or via intervening amino acid sequences. Forexample, a CH1 or CK domain will be fused to an Fc domain (or CH2 or CH3domain thereof) via a linker peptide, optionally a hinge region or afragment thereof. In another example, a VH or VK domain will be fused toa CH3 domain via a linker peptide. VH and VL domains linked to anotherin tandem will be fused via a linker peptide (e.g. as an scFv). VH andVL domains linked to an Fc domain will be fused via a linker peptide.Two polypeptide chains will be bound to one another (indicated by“_(|)”), preferably by interchain disulfide bonds formed betweencysteine residues within complementary CH1 and CK domains.

Linkers for Variable Domains

In one embodiment, a peptide linker for use in linking an ABD (e.g. anscFv, a VH or VL domain) to a CH2 or CH3 comprises a fragment of a CH1domain. For example, a N-terminal amino acid sequence of CH1 can befused to an ABD (e.g. an scFv, a VH or VL domain, etc.) in order tomimic as closely as possible the natural structure of an antibody. Inone embodiment, the linker may comprise a N-terminal CH1 amino acidsequence of between 2-4 residues, between 2-4 residues, between 2-6residues, between 2-8 residues, between 2-10 residues, between 2-12residues, between 2-14 residues, between 2-16 residues, between 2-18residues, between 2-20 residues, between 2-22 residues, between 2-24residues, between 2-26 residues, between 2-28 residues, or between 2-30residues. In one embodiment linker comprises or consists of the aminoacid sequence RTVA.

When an ABD is an scFv, the VH domain and VL domains (VL or VH domainsor fragments thereof that retain binding specificity) that form a scFvare linked together by a linker of sufficient length to enable the ABDto fold in such a way as to permit binding to the antigen for which theABD is intended to bind. Examples of linkers include, for example,linkers comprising glycine and serine residues, e.g., the amino acidsequence GEGTSTGS(G₂S)₂GGAD. In another specific embodiment, the VHdomain and VL domains of an svFv are linked together by the amino acidsequence (G₄S)₃.

Any of the peptide linkers may comprise a length of at least 5 residues,at least 10 residues, at least 15 residues, at least 20 residues, atleast 25 residues, at least 30 residues or more. In other embodiments,the linkers comprises a length of between 2-4 residues, between 2-4residues, between 2-6 residues, between 2-8 residues, between 2-10residues, between 2-12 residues, between 2-14 residues, between 2-16residues, between 2-18 residues, between 2-20 residues, between 2-22residues, between 2-24 residues, between 2-26 residues, between 2-28residues, or between 2-30 residues.

In one embodiment, the hinge region will be a fragment of a hinge region(e.g. a truncated hinge region without cysteine residues) or maycomprise one or amino acid modifications to remove (e.g. substitute byanother amino acid, or delete) a cysteine residue, optionally bothcysteine residues in a hinge region. Removing cysteines can be useful toprevent formation of disulfide bridges in a monomeric polypeptide.

Constant Regions

Constant region domains can be derived from any suitable antibody. Ofparticular interest are the heavy chain domains, including, the constantheavy (CH) domains and the hinge domains. In the context of IgGantibodies, the IgG isotypes each have three CH regions. Accordingly,“CH” domains in the context of IgG are as follows: “CH1” refers topositions 118-220 according to the EU index as in Kabat. “CH2” refers topositions 237-340 according to the EU index as in Kabat, and “CH3”refers to positions 341-447 according to the EU index as in Kabat. By“hinge” or “hinge region” or “antibody hinge region” is meant theflexible polypeptide comprising the amino acids between the first andsecond constant domains of an antibody. Structurally, the IgG CH1 domainends at EU position 220, and the IgG CH2 domain begins at residue EUposition 237. Thus for IgG the hinge is herein defined to includepositions 221 (D221 in IgG1) to 236 (G236 in IgG1), wherein thenumbering is according to the EU index as in Kabat. References to aminoacid residue within constant region domains found within thepolypeptides shall be, unless otherwise indicated or as otherwisedictated by context, with reference to Kabat, in the context of an IgGantibody.

CH3 domains that can serve in the present antibodies can be derived fromany suitable antibody. Such CH3 domains can serve as the basis for amodified CH3 domain. Optionally the CH3 domain is of human origin.

In certain embodiments herein (e.g. for monomeric, dimeric or trimericbispecific antibodies with monomeric Fc domains), a CH3 domain maycomprise one or more amino acid modifications (e.g. amino acidsubstitutions) to disrupt the CH3 dimerization interface. Optionally theCH3 domain modifications will prevent protein aggregation caused by theexposure of hydrophobic residues when the CH2-CH3 domains are inmonomeric form. Optionally, the CH3 domain modifications willadditionally not abolish the ability of the Fc-derived polypeptide tobind to neonatal Fc receptor (FcRn), e.g. human FcRn.

CH3 domains that can be used to prevent homodimer formation have beendescribed in various publications. See, e.g. US 2006/0074225,WO2006/031994, WO2011/063348 and Ying et al. (2012) J. Biol. Chem.287(23):19399-19407, the disclosures of each of which are incorporatedherein by reference. In order to discourage homodimer formation, one ormore residues that make up the CH3-CH3 interface are replaced with acharged amino acid such that the interaction becomes electrostaticallyunfavorable. For example, WO2011/063348 provides that a positive-chargedamino acid in the interface, such as lysine, arginine, or histidine, isreplaced with a different (e.g. negative-charged amino acid, such asaspartic acid or glutamic acid), and/or a negative-charged amino acid inthe interface is replaced with a different (e.g. positive charged) aminoacid. Using human IgG as an example, charged residues within theinterface that may be changed to the opposing charge include R355, D356,E357, K370, K392, D399, K409, and K439. In certain embodiments, two ormore charged residues within the interface are changed to an oppositecharge. Exemplary molecules include those comprising K392D and K409Dmutations and those comprising D399K and D356K mutations. In order tomaintain stability of the polypeptide in monomeric form, one or morelarge hydrophobic residues that make up the CH3-CH3 interface arereplaced with a small polar amino acid. Using human IgG as an example,large hydrophobic residues of the CH3-CH3 interface include Y349, L351,L368, L398, V397, F405, and Y407. Small polar amino acid residuesinclude asparagine, cysteine, glutamine, serine, and threonine. Thus inone embodiment, a CH3 domain will comprise an amino acid modification(e.g. substitution) at 1, 2, 3, 4, 5, 6, 7 or 8 of the positions R355,D356, E357, K370, K392, D399, K409, and K439. In WO2011/063348, two ofthe positively charged Lys residues that are closely located at the CH3domain interface were mutated to Asp. Threonine scanning mutagenesis wasthen carried out on the structurally conserved large hydrophobicresidues in the background of these two Lys to Asp mutations. Fcmolecules comprising K392D and K409D mutations along with the varioussubstitutions with threonine were analyzed for monomer formation.Exemplary monomeric Fc molecules include those having K392D, K409D andY349T substitutions and those having K392D, K409D and F405Tsubstitutions.

In Ying et al. (2012) J. Biol. Chem. 287(23):19399-19407, amino acidsubstitutions were made within the CH3 domain at residues L351, T366,L368, P395, F405, T407 and K409. Combinations of different mutationsresulted in the disruption of the CH3 dimerization interface, withoutcausing protein aggregation. Thus in one embodiment, a CH3 domain willcomprise an amino acid modification (e.g. substitution) at 1, 2, 3, 4,5, 6 or 7 of the positions L351, T366, L368, P395, F405, T407 and/orK409. In one embodiment, a CH3 domain will comprise amino acidmodifications L351Y, T366Y, L368A, P395R, F405R, T407M and K409A. In oneembodiment, a CH3 domain will comprise amino acid modifications L351S,T366R, L368H, P395K, F405E, T407K and K409A. In one embodiment, a CH3domain will comprise amino acid modifications L351 K, T366S, P395V,F405R, T407A and K409Y.

In one embodiment a CH2-CH3 portion comprising a CH3 domain modified toprevent homodimer formation comprises an amino acid sequence of SEQ IDNO: 2, or a sequence at least 90, 95% or 98% identical thereto:APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 2), optionally comprisinga substitution at 1, 2, 3, 4, 5, 6 of residues 121, 136, 165, 175, 177or 179 of SEQ ID NO: 2.

In certain embodiments herein for monomeric, dimeric or trimericbispecific antibodies with monomeric Fc domains, an Fc domain comprisesa tandem CH3 domain. A tandem CH3 domain comprises a first CH3 domain isconnected to a second CH3 domain by a linker. The tandem CH3 domains canthus be placed on a polypeptide chain so as to have the domainarrangement, from N-terminus to C-terminus, as follows:

—CH3-linker-CH3-.

The linker will be a flexible linker (e.g. peptide linker). In oneembodiment the linker permits the CH3 domains to associate with oneanother by non-covalent interactions. In one embodiment, the linker is apeptide linker having 10-50 amino acid residues. In one embodiment, thelinker has the formula (G₄S)_(x). Optionally, x is 2, 3, 4, 5 or 6. Inany of the embodiments, each CH3 domain is independently a full-lengthand/or native CH3 domain, or a fragment or modified CH3 domain whichretains a functional CH3 dimerization interface.

An exemplary tandem CH3 with a flexible peptide linker (underlined) isshown below. An exemplary tandem CH3 domain can thus comprise an aminoacid sequence of SEQ ID NO: 112, or a sequence at least 70%, 80%, 90%,95% or 98% identical thereto:

(SEQ ID NO: 112) G Q P R E P Q V Y T L P P S R E E M T K N Q V S L TC L V K G F Y P S D I A V E W E S N G Q P E N N Y KT T P P V L D S D G S F F L Y S K L T V D K S R W QQ G N V F S C S V M H E A L H N H Y T Q K S L S L S P G  G   G   G   G  S   G   G   G   G   S   G   G   G   G S  G Q P R E P Q V YT L P P S R E E M T K N Q V S L T C L V K G F Y P SD I A V E W E S N G Q P E N N Y K T T P P V L D S DG S F F L Y S K L T V D K S R W Q Q G N V F S C S VM H E A L H N H Y T Q K S L S L S P G

CH2 domains can be readily obtained from any suitable antibody.Optionally the CH2 domain is of human origin. A CH2 may or may not belinked (e.g. at its N-terminus) to a hinge of linker amino acidsequence. In one embodiment, a CH2 domain is a naturally occurring humanCH2 domain of IgG1, 2, 4 or 4 subclass. In one embodiment, a CH2 domainis a fragment of a CH2 domain (e.g. at least 10, 20, 30, 40 or 50 aminoacids).

In one embodiment, a CH2 domain, when present in a polypeptide describedherein, will retain binding to a neonatal Fc receptor (FcRn),particularly human FcRn.

In one embodiment, a CH2 domain, when present in a polypeptide describedherein, and the polypeptides described herein, will confer decreased orlack of binding to a Fcγ receptor, notably FcγRIIIA (CD16).

In one embodiment, the polypeptides described herein and their Fcdomain(s) and/or a CH2 domain thereof, will have decreased or willsubstantially lack antibody dependent cytotoxicity (ADCC), complementdependent cytotoxicity (CDC), antibody dependent cellular phagocytosis(ADCP), FcR-mediated cellular activation (e.g. cytokine release throughFcR cross-linking), and/or FcR-mediated platelet activation/depletion,as mediated by NKp46-negative immune cells.

In one embodiment, a CH2 domain in a polypeptide will have substantialloss of binding to activating Fcγ receptors, e.g., FcγRIIIA (CD16),FcγRIIA (CD32A) or CD64, or to an inhibitory Fc receptor, e.g., FcγRIIB(CD32B). In one embodiment, a CH2 domain in a polypeptide willfurthermore have substantial loss of binding to the first component ofcomplement (C1q).

The exemplary multispecific proteins described herein make use ofwild-type CH2 domains in monomeric Fc domains, or with CH2 mutations indimeric Fc domain proteins at reside N297 (Kabat numbering). However theperson of skill in the art will appreciate that other configurations canbe implemented. For example, substitutions into human IgG1 of IgG2residues at positions 233-236 and IgG4 residues at positions 327, 330and 331 were shown to greatly reduce binding to Fcγ receptors and thusADCC and CDC. Furthermore, Idusogie et al. (2000) J Immunol.164(8):4178-84 demonstrated that alanine substitution at differentpositions, including K322, significantly reduced complement activation.

In one embodiment, a CH2 domain that retains binding to a FcRn receptorbut has reduction of binding to Fcγ receptors will lack or have modifiedN-linked glycosylation, e.g. at residue N297 (Kabat EU). For example thepolypeptide is expressed in a cell line which naturally has a highenzyme activity for adding fucosyl to the N-acetylglucosamine that bindsto the Fc region of the polypeptides, or which does not yieldglycosylation at N297 (e.g. bacterial host cells). In anotherembodiment, a polypeptide may have one or more substitution that resultin lack of the canonical Asn-X-Ser/Thr N-linked glycosylation motif atresidues 297-299, which can also thus also result in reduction ofbinding to Fcγ receptors. Thus, a CH2 domain may have a substitution atN297 and/or at neighboring residues (e.g. 298, 299).

In one embodiment, an Fc domain or a CH2 domain therefrom is derivedfrom an IgG1, IgG3, IgG4 or IgG2 Fc mutant exhibiting diminished FcγRbinding capacity but having conserved FcRn binding. In one aspect, theIgG2 Fc mutant or the derived multispecific polypeptide, Fc domain orCH2 domain comprises the mutations V234A, G237A, P238S according to theEU numbering system. In another aspect, the IgG2 Fc mutant or thederived multispecific polypeptide or Fc domain comprises mutationsV234A, G237A, H2680 or H268A, V309L, A330S, P331S according to the EUnumbering system. In a particular aspect, the IgG2 Fc mutant or thederived multispecific polypeptide or Fc domain comprises mutationsV234A, G237A, P238S, H268A, V309L, A330S, P331S, and, optionally, P233Saccording to the EU numbering system. Optionally, a CH2 domain with lossof binding to Fcγ receptors may comprises residues 233, 234, 235, 237,and 238 (EU numbering system) that comprise a n amino acid sequenceselected from PAAAP, PAAAS, and SAAAS; optionally an Fc domain havingsuch mutations can further comprise mutations H268A or H268Q, V309L,A330S and P331S (see WO2011/066501, the disclosure of which isincorporated herein by reference).

In one embodiment, a CH2 domain that loses binding to a Fcγ receptorwill comprise at least one amino acid modification (for example,possessing 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications)in the CH2 domain of the Fc region, optionally further in combinationwith one or more amino acid modification in other domains (e.g. in ahinge domain or a CH3 domain). Any combination of Fc modifications canbe made, for example any combination of different modificationsdisclosed in Armour KL. et al., (1999) Eur J Immunol. 29(8):2613-24;Presta, L. G. et al. (2002) Biochem. Soc. Trans. 30(4):487-490; Shields,R. L. et al. (2002) J. Biol. Chem. 26; 277(30):26733-26740 and Shields,R. L. et al. (2001) J. Biol. Chem. 276(9):6591-6604). In one embodiment,a polypeptide of the invention that has decreased binding to a human Fcγreceptor will comprise at least one amino acid modification (forexample, possessing 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acidmodifications) relative to a wild-type CH2 domain within amino acidresidues 237-340 (EU numbering), such that the polypeptide comprisingsuch CH2 domain has decreased affinity for a human Fcγ receptor ofinterest relative to an equivalent polypeptide comprising a wild-typeCH2 domain, optionally wherein the variant CH2 domain comprises asubstitution at any one or more of positions 233, 234, 235, 236, 237,238, 268, 297, 238, 299, 309, 327, 330, 331 (EU numbering).

CDR Sequences and Epitopes

In one embodiment, the proteins and antibodies herein bind the D1 domainof NKp46, the D2 domain of NKp46, or to a region spanning both the D1and D2 domains (at the border of the D1 and D2 domains, the D1/D2junction), of the NKp46 polypeptide of SEQ ID NO: 1. In one embodiment,the proteins or antibodies have an affinity for human NKp46characterized by a K_(D) of less than 10⁻⁸ M, less than 10⁻⁹ M, or lessthan 10⁻¹⁰M.

In another embodiment, the antibodies bind NKp46 at substantially thesame epitope on NKp46 as antibody NKp46-1, NKp46-2, NKp46-3, NKp46-4,NKp46-6 or NKp46-. In another embodiment, the antibodies at leastpartially overlaps, or includes at least one residue in the segmentbound by NKp46-1, NKp46-2, NKp46-3, NKp46-4, NKp46-6 or NKp46. In oneembodiment, all key residues of the epitope are in a segmentcorresponding to domain D1 or D2. In one embodiment, the antibody bindsa residue present in the D1 domain as well as a residue present in inthe D2 domain. In one embodiment, the antibodies bind an epitopecomprising 1, 2, 3, 4, 5, 6, 7 or more residues in the segmentcorresponding to domain D1 or D2 of the NKp46 polypeptide of SEQ IDNO: 1. In one embodiment, the antibodies bind domain D1 and bind anepitope comprising 1, 2, 3, or 4 of the residues R101, V102, E104 and/orL105.

In one embodiment, the antibodies bind domain D1/D2 junction and bind anepitope comprising 1, 2, 3, 4 or 5 of the residues K41, E42, E119, Y121and/or Y194.

In one embodiment, the antibodies bind domain D2 and bind an epitopecomprising 1, 2, 3, or 4 of the residues P132, E133, 1135, and/or S136.

The Examples section herein describes the construction of a series ofmutant human NKp46 polypeptides. Binding of anti-NKp46 antibody to cellstransfected with the NKp46 mutants was measured and compared to theability of anti-NKp46 antibody to bind wild-type NKp46 polypeptide (SEQID NO:1). A reduction in binding between an anti-NKp46 antibody and amutant NKp46 polypeptide as used herein means that there is a reductionin binding affinity (e.g., as measured by known methods such FACStesting of cells expressing a particular mutant, or by Biacore testingof binding to mutant polypeptides) and/or a reduction in the totalbinding capacity of the anti-NKp46 antibody (e.g., as evidenced by adecrease in Bmax in a plot of anti-NKp46 antibody concentration versuspolypeptide concentration). A significant reduction in binding indicatesthat the mutated residue is directly involved in binding to theanti-NKp46 antibody or is in close proximity to the binding protein whenthe anti-NKp46 antibody is bound to NKp46. An antibody epitope will thuspreferably include such residue and may include additional residuesadjacent to such residue.

In some embodiments, a significant reduction in binding means that thebinding affinity and/or capacity between an anti-NKp46 antibody and amutant NKp46 polypeptide is reduced by greater than 40%, greater than50%, greater than 55%, greater than 60%, greater than 65%, greater than70%, greater than 75%, greater than 80%, greater than 85%, greater than90% or greater than 95% relative to binding between the antibody and awild type NKp46 polypeptide (e.g., the polypeptide shown in SEQ IDNO:1). In certain embodiments, binding is reduced below detectablelimits. In some embodiments, a significant reduction in binding isevidenced when binding of an anti-NKp46 antibody to a mutant NKp46polypeptide is less than 50% (e.g., less than 45%, 40%, 35%, 30%, 25%,20%, 15% or 10%) of the binding observed between the anti-NKp46 antibodyand a wild-type NKp46 polypeptide (e.g., the polypeptide shown in SEQ IDNO: 1 (or the extracellular domain thereof)). Such binding measurementscan be made using a variety of binding assays known in the art. Aspecific example of one such assay is described in the Example section.

In some embodiments, anti-NKp46 antibodies are provided that exhibitsignificantly lower binding for a mutant NKp46 polypeptide in which aresidue in a wild-type NKp46 polypeptide (e.g., SEQ ID NO:1) issubstituted. In the shorthand notation used here, the format is: Wildtype residue: Position in polypeptide: Mutant residue, with thenumbering of the residues as indicated in SEQ ID NO: 1.

In some embodiments, an anti-NKp46 antibody binds a wild-type NKp46polypeptide but has decreased binding to a mutant NKp46 polypeptidehaving a mutation (e.g., an alanine substitution) any one or more of theresidues R101, V102, E104 and/or L105 (with reference to SEQ ID NO:1)compared to binding to the wild-type NKp46).

In some embodiments, an anti-NKp46 antibody binds a wild-type NKp46polypeptide but has decreased binding to a mutant NKp46 polypeptidehaving a mutation (e.g., an alanine substitution) any one or more of theresidues K41, E42, E119, Y121 and/or Y194 (with reference to SEQ IDNO:1) compared to binding to the wild-type NKp46).

In some embodiments, an anti-NKp46 antibody binds a wild-type NKp46polypeptide but has decreased binding to a mutant NKp46 polypeptidehaving a mutation (e.g., an alanine substitution) any one or more of theresidues P132, E133, 1135, and/or S136 (with reference to SEQ ID NO:1)compared to binding to the wild-type NKp46)

The amino acid sequence of the heavy chain variable region of antibodiesNKp46-1, NKp46-2, NKp46-3, NKp46-4, NKp46-6 and NKp46-9 are listedherein in Table B (SEQ ID NOS: 3, 5, 7, 9, 11 and 13 respectively), theamino acid sequence of the light chain variable region of antibodiesNKp46-1, NKp46-2, NKp46-3, NKp46-4, NKp46-6 and NKp46-9 are also listedherein in Table B (SEQ ID NOS: 4, 6, 8, 10, 12 and 14 respectively).

In a specific embodiment, provided is an antibody, e.g. a full lengthmonospecific antibody, a multispecific or bispecific antibody, includinga bispecific monomeric polypeptide, that binds essentially the sameepitope or determinant as monoclonal antibody NKp46-1, NKp46-2, NKp46-3,NKp46-4, NKp46-6 or NKp46-9; optionally the antibody comprises ahypervariable region of antibody NKp46-1, NKp46-2, NKp46-3, NKp46-4,NKp46-6 or NKp46-9. In any of the embodiments herein, antibody NKp46-1,NKp46-2, NKp46-3, NKp46-4, NKp46-6 or NKp46-9 can be characterized byits amino acid sequence and/or nucleic acid sequence encoding it. In oneembodiment, the antibody comprises the Fab or F(ab′)₂ portion ofNKp46-1, NKp46-2, NKp46-3, NKp46-4, NKp46-6 or NKp46-9. Also provided isan antibody that comprises the heavy chain variable region of NKp46-1,NKp46-2, NKp46-3, NKp46-4, NKp46-6 or NKp46-9. According to oneembodiment, an antibody comprises the three CDRs of the heavy chainvariable region of NKp46-1, NKp46-2, NKp46-3, NKp46-4, NKp46-6 orNKp46-9. Also provided is a polypeptide that further comprises one, twoor three of the CDRs of the light chain variable region of NKp46-1,NKp46-2, NKp46-3, NKp46-4, NKp46-6 or NKp46-9. Optionally any one ormore of said light or heavy chain CDRs may contain one, two, three, fouror five or more amino acid modifications (e.g. substitutions, insertionsor deletions). Optionally, provided is a polypeptide where any of thelight and/or heavy chain variable regions comprising part or all of anantigen binding region of antibody NKp46-1, NKp46-2, NKp46-3, NKp46-4,NKp46-6 or NKp46-9 are fused to an immunoglobulin constant region of thehuman IgG type.

In another aspect, the invention provides a protein, e.g., an antibody,a full length monospecific antibody, a multispecific or a bispecificprotein, or a polypeptide chain or fragment thereof, as well as anucleic acid encoding any of the foregoing, wherein the proteincomprises the heavy chain CDRs of NKp46-1, NKp46-2, NKp46-3, NKp46-4,NKp46-6 or NKp46-9, comprising, for the respective antibody: a HCDR1region comprising an amino acid sequence as set forth in Table A, or asequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acidsthereof, wherein one or more of these amino acids may be substituted bya different amino acid; a HCDR2 region comprising an amino acid sequenceas set forth in Table A, or a sequence of at least 4, 5, 6, 7, 8, 9 or10 contiguous amino acids thereof, wherein one or more of these aminoacids may be substituted by a different amino acid; a HCDR3 regioncomprising an amino acid sequence as set forth in as set forth in TableA, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous aminoacids thereof, wherein one or more of these amino acids may besubstituted by a different amino acid.

In another aspect, the invention provides a protein, e.g., an antibody,a full length monospecific antibody, a multispecific or a bispecificprotein, or a polypeptide chain or fragment thereof, as well as anucleic acid encoding any of the foregoing, wherein the proteincomprises light chain CDRs of NKp46-1, NKp46-2, NKp46-3, NKp46-4,NKp46-6 or NKp46-9, comprising, for the respective antibody: a LCDR1region comprising an amino acid sequence as set forth in Table A, or asequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acidsthereof, wherein one or more of these amino acids may be substituted bya different amino acid; a LCDR2 region comprising an amino acid sequenceas set forth in Table A, or a sequence of at least 4, 5, 6, 7, 8, 9 or10 contiguous amino acids thereof, wherein one or more of these aminoacids may be substituted by a different amino acid; a LCDR3 regioncomprising an amino acid sequence as set forth in Table A, or a sequenceof at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof,wherein one or more of these amino acids may be deleted or substitutedby a different amino acid.

In another aspect, the invention provides a protein that binds humanNKp46, comprising:

(a) the heavy chain variable region of NKp46-1, NKp46-2, NKp46-3,NKp46-4, NKp46-6 or NKp46-9 as set forth in Table B, optionally whereinone, two, three or more amino acids may be substituted by a differentamino acid;(b) the light chain variable region NKp46-1, NKp46-2, NKp46-3, NKp46-4,NKp46-6 or NKp46-9 as set forth in Table B, optionally wherein one, two,three or more amino acids may be substituted by a different amino acid;(c) the heavy chain variable region of NKp46-1, NKp46-2, NKp46-3,NKp46-4, NKp46-6 or NKp46-9 as set forth in Table B, optionally whereinone or more of these amino acids may be substituted by a different aminoacid; and the respective light chain variable region of NKp46-1,NKp46-2, NKp46-3, NKp46-4, NKp46-6 or NKp46-9 as set forth in Table B,optionally wherein one, two, three or more amino acids may besubstituted by a different amino acid;(d) the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2) amino acid sequence ofNKp46-1, NKp46-2, NKp46-3, NKp46-4, NKp46-6 or NKp46-9 as shown in TableA, optionally wherein one, two, three or more amino acids in a CDR maybe substituted by a different amino acid;(e) the light chain CDR 1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acidsequence of NKp46-1, NKp46-2, NKp46-3, NKp46-4, NKp46-6 or NKp46-9 asshown in Table A, optionally wherein one, two, three or more amino acidsin a CDR may be substituted by a different amino acid; or(f) the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acidsequence of NKp46-1, NKp46-2, NKp46-3, NKp46-4, NKp46-6 or NKp46-9 asshown in Table A, optionally wherein one, two, three or more amino acidsin a CDR may be substituted by a different amino acid; and the lightchain CDRs 1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acid sequence of therespective NKp46-1, NKp46-2, NKp46-3, NKp46-4, NKp46-6 or NKp46-9antibody as shown in Table A, optionally wherein one, two, three or moreamino acids in a CDR may be substituted by a different amino acid.

In one embodiment, the aforementioned CDRs are according to Kabat, e.g.as shown in Table A. In one embodiment, the aforementioned CDRs areaccording to Chotia numbering, e.g. as shown in Table A. In oneembodiment, the aforementioned CDRs are according to IMGT numbering,e.g. as shown in Table A.

In another aspect of any of the embodiments herein, any of the CDRs 1, 2and 3 of the heavy and light chains may be characterized by a sequenceof at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof,and/or as having an amino acid sequence that shares at least 50%, 60%,70%, 80%, 85%, 90% or 95% sequence identity with the particular CDR orset of CDRs listed in the corresponding SEQ ID NO or Table A.

In another aspect, the invention provides an antibody that competes forNKp46 binding with a monoclonal antibody of (a) to (f), above.

In another aspect, the invention provides a bispecific antibodycomprising an antibody that binds human NKp46 of (a) to (f), above, oran antibody that competes for binding to NKp46 therewith, fused(optionally via intervening amino acid sequences) to a monomeric Fcdomain, optionally further fused (optionally via intervening amino acidsequences) to a second antigen binding domain (e.g. a scFv, a V_(H)domain, a V_(L) domain, a dAb, a V-NAR domain or a V_(H)H domain).Optionally the second antigen binding domain will bind a cancer antigen,a viral antigen or a bacterial antigen.

The sequences of the CDRs, according to IMGT, Kabat and Chothiadefinitions systems, have been summarized in Table A below. Thesequences of the variable chains of the antibodies according to theinvention are listed in Table B below. In any embodiment herein, a VL orVH sequence can be specified or numbered so as to contain or lack asignal peptide or any part thereof.

TABLE A HCDR1 HCDR2 HCDR3 CDR SEQ SEQ SEQ mAb definition ID Sequence IDSequence ID Sequence NKp46-1 Kabat 15 DYVIN 18 EIYPGSGTNYYNEKFKA 21RGRYGLYAMDY Chotia 16 GYTFTDY 19 PGSG 22 GRYGLYAMD IMGT 17 GYTFTDYV 20GYTFTDYVIYPGSGTN 23 ARRGRYGLYAMDY NKp46-2 Kabat 31 SDYAWN 34YITYSGSTSYNPSLES 36 GGYYGSSWGVFAY Chotia 32 GYSITSDY YSG 37 GYYGSSWGVFAIMGT 33 GYSITSDYA 35 ITYSGST 38 ARGGYYGSSWG VFAY NKp46-3 Kabat 46 EYTMH49 GISPNIGGTSYNQKFKG 51 RGGSFDY Chotia 47 GYTFTEY PNIG 52 GGSFD IMGT 48GYTFTEYT 50 ISPNIGGT 53 ARRGGSFDY NKp46-4 Kabat 60 SFTMH 63YINPSSGYTEYNQKFKD 65 GSSRGFDY Chotia 61 GYTFTSF PSSG 66 SSRGFD IMGT 62GYTFTSFT 64 INPSSGYT 67 VRGSSRGFDY NKp46-6 Kabat 73 SSWMH 76HIHPNSGISNYNEKFKG 78 GGRFDD Chotia 74 GYTFTSS PNSG GRFD IMGT 75 GYTFTSSW77 IHPNSGIS 79 ARGGRFDD NKp46-9 Kabat 85 SDYAWN 88 YITYSGSTNYNPSLKS 89CWDYALYAMDC Chotia 86 GYSITSDY YSG 90 WDYALYAMD IMGT 87 GYSITSDYA 35ITYSGST 91 ARCWDYALYAMDC Bab281 Kabat 97 NYGMN 100 WINTNTGEPTYAEEFKG 102DYLYYFDY Chotia 98 GYTFTNY TNTG 103 YLYYFD IMGT 99 GYTFTNYG 101 INTNTGEP104 ARDYLYYFDY LCDR1 LCDR2 LCDR3 CDR SEQ SEQ SEQ mAb definition IDSequence ID Sequence ID Sequence NKp46-1 Kabat 24 RASQDISNYLN 27 YTSRLHS28 QQGNTRPWT Chotia 25 SQDISNY YTS 29 YTSGNTRPW IMGT 26 QDISNY YTS 30YTSQQGNTRPWT NKp46-2 Kabat 39 RVSENIYSYLA 42 NAKTLAE 43 QHHYGTPWT Chotia40 SENIYSY NAK 44 HYGTPW IMGT 41 ENIYSY NAK 45 QHHYGTPWT NKp46-3 Kabat54 RASQSISDYLH 57 YASQSIS 58 QNGHSFPLT Chotia 55 SQSISDY YAS 59 GHSFPLIMGT 56 QSISDY YAS QNGHSFPLT NKp46-4 Kabat 68 RASENIYSNLA 70 AATNLAD 71QHFWGTPRT Chotia SENIYSN AAT 72 FWGTPR IMGT 69 ENIYSN AAT QHFWGTPRTNKp46-6 Kabat 80 RASQSISDYLH YASQSIS 82 QNGHSFLMYT Chotia 81 GRFDSQSISDYYAS 83 GHSFLMY IMGT QSISDY YAS 84 YASQNGHSFLMYT NKp46-9 Kabat 92RTSENIYSYLA 93 NAKTLAE 94 QHHYDTPLT Chotia SENIYSY NAK 95 NAKHYDTPL IMGTENIYSY NAK 96 QHHYDTPLT Bab281 Kabat 105 KASENVVTYVS 108 GASNRYT 109GQGYSYPYT Chotia 106 SENVVTY GAS 110 GYSYPY IMGT 107 ENVVTY GAS 111GQGYSYPYT

TABLE B SEQ ID Antibody NO Amino acid sequence NKp46-1 VH 3QVQLQQSGPELVKPGASVKMSCKASGYTFTDYVINWGKQRSGQGLEWIGEIYPGSGTNYYNEKFKAKATLTADKSSNIAYMQLSSLTSEDSAVYFCARRGRY GLYAMDYWGQGTSVTVSSNKp46-1 VL 4 DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTINNLEQEDIATYFCQQGNTRPWTFGGGT KLEIK NKp46-2 VH 5EVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYITYSGSTSYNPSLESRISITRDTSTNQFFLQLNSVTTEDTATYYCARGGYY GSSWGVFAYWGQGTLVTVSANKp46-2 VL 6 DIQMTQSPASLSASVGETVTITCRVSENIYSYLAWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDEGSYYCQHHYGTPWTEGGGT KLEIK NKp46-3 VH 7EVQLQQSGPELVKPGASVKISCKTSGYTFTEYTMHWVKQSHGKSLEWIGGISPNIGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRGGS FDYWGQGTTLTVSSNKp46-3 VL 8 DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGT KLELK NKp46-4 VH 9QVQLQQSAVELARPGASVKMSCKASGYTFTSFTMHWVKQRPGQGLEWIGYINPSSGYTEYNQKFKDKTTLTADKSSSTAYMQLDSLTSDDSAVYYCVRGSSR GFDYWGQGTLVTVSANKp46-4 VL 10 DIQMIQSPASLSVSVGETVTITCRASENIYSNLAWFQQKQGKSPQLLVYAATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGIYYCQHFWGTPRTFGGGT KLEIK NKp46-6 VH 11QVQLQQPGSVLVRPGASVKLSCKASGYTFTSSWMHWAKQRPGQGLEWIGHIHPNSGISNYNEKFKGKATLTVDTSSSTAYVDLSSLTSEDSAVYYCARGGRF DDWGAGTTVTVSSNKp46-6 VL 12 DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFLMYTEGGG TKLEIK NKp46-9 VH 13DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYITYSGSTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARCWDY ALYAMDCWGQGTSVTVSSNKp46-9 VL 14 DIQMTQSPASLSASVGETVTITCRTSENIYSYLAWCQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTHFSLKINSLQPEDFGIYYCQHHYDTPLTFGAGT KLELK

Also provided, as shown in the Examples herein, is a protein comprisingthe amino acid sequences of monomeric bispecific polypeptides comprisingscFv comprising the heavy and light chain CDR1, 2 and 3 of therespective heavy and light chain variable region listed as SEQ ID NOS:3-14 of antibodies NKp46-1, NKp46-2, NKp46-3, NKp46-4, NKp46-6 andNKp46-9, a monomeric Fc domain, and scFv comprising the heavy and lightchain CDR1, 2 and 3 of the heavy and light chain variable region of ananti-CD19 antibodies, e.g. the anti-CD19 shown in the Example herein.

Once the multispecific protein is produced it can be assessed forbiological activity, such as agonist activity.

In one aspect of any embodiment herein, a multispecific protein iscapable of inducing activation of an NKp46-expressing cell (e.g. an NKcell, a reporter cell) when the protein is incubated in the presence ofthe NKp46-expressing cell (e.g. purified NK cells) and a target cellthat expresses the antigen of interest).

In one aspect of any embodiment herein, a multispecific protein isincapable of inducing substantial activation of an NKp46-expressing cell(e.g. an NK cell, a reporter cell) when incubated with NKp46-expressingcells (e.g., purified NK cells or purified reporter cells, optionallyfurther in the presence of Fcγ receptor-expressing cells) in the absenceof target cells.

In one aspect of any embodiment herein, a multispecific protein iscapable of inducing NKp46 signaling in an NKp46-expressing cell (e.g. anNK cell, a reporter cell) when the protein is incubated in the presenceof the NKp46-expressing cell (e.g. purified NK cells) and a target cellthat expresses the antigen of interest).

In one aspect of any embodiment herein, a multispecific protein is notcapable of causing (or increasing) NKp46 signaling in anNKp46-expressing cell (e.g. an NK cell, a reporter cell) when incubatedwith NKp46-expressing cells (e.g., purified NK cells or purifiedreporter cells, optionally further in the presence of Fcγreceptor-expressing cells) in the absence of target cells.

Optionally, NK cell activation or signaling in characterized byincreased expression of a cell surface marker of activation, e.g. CD107,CD69, etc.

Activity can be measured for example by bringing target cells andNKp46-expressing cells into contact with one another, in presence of themultispecific polypeptide. In one example, aggregation of target cellsand NK cells is measured. In another example, the multispecific proteinmay, for example, be assessed for the ability to cause a measurableincrease in any property or activity known in the art as associated withNK cell activity, respectively, such as marker of cytotoxicity (CD107)or cytokine production (for example IFN-γ or TNF-α), increases inintracellular free calcium levels, the ability to lyse target cells in aredirected killing assay, etc.

In the presence of target cells (target cells expressing the antigen ofinterest) and NK cells that express NKp46, the multispecific proteinwill be capable of causing an increase in a property or activityassociated with NK cell activity (e.g. activation of NK cellcytotoxicity, CD107 expression, IFNγ production) in vitro. For example,an multispecific protein of the disclosure can be selected for theability to increase an NK cell activity by more than about 20%,preferably with at least about 30%, at least about 40%, at least about50%, or more compared to that achieved with the same effector: targetcell ratio with the same NK cells and target cells that are not broughtinto contact with the multispecific protein, as measured by an assay ofNK cell activity, e.g., a marker of activation of NK cell cytotoxicity,CD107 or CD69 expression, IFNγ production, a classical in vitro chromiumrelease test of cytotoxicity. Examples of protocols for activation andcytotoxicity assays are described in the Examples herein, as well as forexample, in Pessino et al, J. Exp. Med, 1998, 188 (5): 953-960; Sivoriet al, Eur J Immunol, 1999. 29:1656-1666; Brando et al, (2005) J.Leukoc. Biol. 78:359-371; EI-Sherbiny et al, (2007) Cancer Research67(18):8444-9; and Nolte-'t Hoen et al, (2007) Blood 109:670-673).

Activity can also be assed using a reporter assay can be used in whichNKp46 ligand-expressing target cells are brought into contact with aNKp46 expressing reporter cell (e.g. an NK cell, a T cell), and theability of the antibody to induce NKp46 signaling is assessed. Forexample, the NKp46-expressing reporter cell may be the DO.11.10 T cellhybridoma or similar cell transduced with retroviral particles encodinga chimeric NKp46 protein in which the intracytoplasmic domain of mouseCD3ζ is fused to the extracellular portion of NKp46 (see, e.g., DOMSP46cells as described in Schleinitz et al., (2008) Arthritis Rheum. 58:3216-3223). Engagement of the chimeric proteins at the cell surfacetriggers IL-2 secretion. After incubation, cell supernatants can beassayed for the presence of mouse IL-2 in a standard target cellsurvival assay. A target cell can be selected that does not, in theabsence of the multispecific protein, induce NKp46 signaling in thereporter cell. The multispecific protein can then be brought intocontact with the NKp46 expressing reporter cell in the presence of thetarget cell, and NKp46 signaling can be assessed. DOMSP46, or DO.11.10(20,000 cells/well in 96-well plates) can be incubated with target cellsand multispecific protein in 96-well plates. After 20 h, cellsupernatants are assayed for the presence of mouse IL-2 in a standardCTLL-2 survival assay using Cell Titer-Glo Luminescent Cell ViabilityAssay (Promega).

In one embodiment, the invention provides methods of making a monomericpolypeptide (e.g. any monomeric protein described herein), comprising:

a) providing a nucleic acid encoding a monomeric bispecific polypeptidedescribed herein (e.g., a polypeptide comprising (a) a first antigenbinding domain that binds to NKp46; (b) a second antigen binding domainthat binds a polypeptide expressed on a target cell; and (c) at least aportion of a human Fc domain, wherein the multispecific polypeptide iscapable of binding to human neonatal Fc receptor (FcRn) and hasdecreased binding to a human Fcγ receptor compared to a full length wildtype human IgG1 antibody); and

b) expressing said nucleic acid in a host cell to produce saidpolypeptide, respectively; and recovering the monomeric protein.Optionally step (b) comprises loading the protein produced onto anaffinity purification support, optionally an affinity exchange column,optionally a Protein-A support or column, and collecting the monomericprotein.

In one embodiment, the invention provides methods of making aheterodimeric protein (e.g. any heterodimeric protein described herein),comprising:

a) providing a first nucleic acid encoding a first polypeptide chaindescribed herein (e.g., a polypeptide chain comprising a first variabledomain (V) fused to a CH1 of CK constant region, a second variabledomain (and optionally third variable domain, wherein the second andthird variable domain form a first antigen binding domain), and an Fcdomain or portion thereof interposed between the first and secondvariable domains);

b) providing a second nucleic acid encoding a second polypeptide chaindescribed herein (e.g., a polypeptide chain comprising a first variabledomain (V) fused at its C-terminus to a CH1 or CK constant regionselected to be complementary to the CH1 or CK constant region of thefirst polypeptide chain such that the first and second polypeptides forma CH1-CK heterodimer in which the first variable domain of the firstpolypeptide chain and the first variable domain of the secondpolypeptide form a second antigen binding domain); wherein one of thefirst or second antigen binding domains binds NKp46 and the other bindsan antigen of interest; and

c) expressing said first and second nucleic acids in a host cell toproduce a protein comprising said first and second polypeptide chains,respectively; and recovering a heterodimeric protein. Optionally, theheterodimeric protein produced represents at least 20%, 25% or 30% ofthe total proteins (e.g. bispecific proteins) prior to purification.Optionally step (c) comprises loading the protein produced onto anaffinity purification support, optionally an affinity exchange column,optionally a Protein-A support or column, and collecting theheterodimeric protein; and/or loading the protein produced (or theprotein collected following loading onto an affinity exchange or ProteinA column) onto an ion exchange column; and collecting the heterodimericfraction. In one embodiment, the second variable domain (optionallytogether with the third variable domain) of the first polypeptide chainbinds NKp46.

By virtue of their ability to be produced in standard cell lines andstandardized methods with high yields, unlike BITE, DART and otherbispecific formats, the proteins of the disclosure also provide aconvenient tool for screening for the most effective variable regions toincorporated into a multispecific protein. In one aspect, the presentdisclosure provides a method for identifying or evaluating candidatevariable regions for use in a heterodimeric protein, comprising thesteps of:

a) providing a plurality of nucleic acid pairs, wherein each pairincludes one nucleic acid encoding a heavy chain candidate variableregion and one nucleic acid encoding a light chain candidate variableregion, for each of a plurality of heavy and light chain variable regionpairs (e.g., obtained from different antibodies binding the same ordifferent antigen(s) of interest);

b) for each of the plurality nucleic acid pairs, making a heterodimericprotein by:

-   -   (i) producing a first nucleic acid encoding a first polypeptide        chain comprising one of the heavy or light chain candidate        variable domains (V) fused to a CH1 or CK constant region, a        second variable domain (and optionally third variable domain,        wherein the second and third variable domain form a first        antigen binding domain), and an Fc domain or portion thereof        interposed between the candidate and second variable domains);    -   (ii) producing a second nucleic acid encoding a second        polypeptide chain comprising the other of the heavy or light        chain candidate variable domains (V) fused at its C-terminus to        a CH1 or CK constant region selected to be complementary to the        CH1 or CK constant region of the first polypeptide chain such        that the first and second polypeptides form a CH1-CK heterodimer        in which the heavy and light chain candidate variable domains        form a second antigen binding domain; and    -   (iii) expressing said nucleic acids encoding the first and        second polypeptide chains in a host cell to produce a protein        comprising said first and second polypeptide chains,        respectively; and recovering a heterodimeric protein; and

c) evaluating the plurality of heterodimeric proteins produced for abiological activity of interest, e.g., an activity disclosed herein. Inthis method, one of the first or second antigen binding domains bindsNKp46 and the other binds an antigen of interest. In one embodiment, thesecond variable domain (optionally together with the third variabledomain) of the first polypeptide chain binds NKp46. Optionally, theheterodimeric protein produced represents at least 20%, 25% or 30% ofthe total proteins prior to purification. Optionally the recovering stepin (iii) comprises loading the protein produced onto an affinitypurification support, optionally an affinity exchange column, optionallya Protein-A support or column, and collecting the heterodimeric protein;and/or loading the protein produced (or the protein collected followingloading onto a affinity exchange or Protein A column) onto an ionexchange column; and collecting the heterodimeric fraction. In oneembodiment, the first antigen binding domain binds NKp46 and the secondantigen binding domain binds an antigen of interest; optionally thefirst antigen binding domain is an anti-NKp46 scFv. In one embodiment,the second variable domain (optionally together with the third variabledomain) of the first polypeptide chain binds NKp46.

In one embodiment, the invention provides methods of making aheterotrimeric protein (e.g. any heterotrimeric protein describedherein), comprising:

(a) providing a first nucleic acid encoding a first polypeptide chaindescribed herein (e.g., a polypeptide chain comprising a first variabledomain (V) fused to a first CH1 or CK constant region, a second variabledomain fused to a second CH1 or CK constant region, and an Fc domain orportion thereof interposed between the first and second (V—CH1/CK)units);

(b) providing a second nucleic acid encoding a second polypeptide chaindescribed herein (e.g., a polypeptide chain comprising a variable domain(V) fused at its C-terminus to a CH1 or CK constant region selected tobe complementary to the first CH1 or CK constant region of the firstpolypeptide chain such that the first and second polypeptides form aCH1-CK heterodimer in which the first variable domain of the firstpolypeptide chain and the variable domain of the second polypeptide forman antigen binding domain);

(c) providing a third nucleic acid comprising a third polypeptide chaindescribed herein (e.g., a polypeptide chain comprising a variable domainfused at its C-terminus to a CH1 or CK constant region, wherein the CH1or CK constant region is selected to be complementary to the secondvariable domain and second CH1 or CK constant region of the firstpolypeptide chain such that the first and third polypeptides form aCH1-CK heterodimer in which the second variable domain of the firstpolypeptide and the variable domain of the third polypeptide form anantigen binding domain; and

(d) expressing said first, second and third nucleic acids in a host cellto produce a protein comprising said first, second and third polypeptidechains, respectively; and recovering a heterotrimeric protein.Optionally, the heterotrimeric protein produced represents at least 20%,25% or 30% of the total proteins prior to purification. Optionally step(d) comprises loading the protein produced onto an affinity purificationsupport, optionally an affinity exchange column, optionally a Protein-Asupport or column, and collecting the heterotrimeric protein; and/orloading the protein produced (e.g., the protein collected followingloading onto an affinity exchange or Protein A column) onto an ionexchange column; and collecting the heterotrimeric fraction. In thismethod, one of the antigen binding domains binds NKp46 and the otherbinds an antigen of interest. In one embodiment, the second or the thirdpolypeptide further comprises and Fc domain or fragment thereof fused tothe C-terminus of the CH1 or CK domain (e.g. via a hinge domain orlinker). In one embodiment, the second variable domain of the firstpolypeptide and the variable domain of the third polypeptide form anantigen binding domain that binds NKp46.

In one aspect, the present disclosure provides a method for identifyingor evaluating candidate variable regions for use in a heterotrimericprotein, comprising the steps of:

a) providing a plurality of nucleic acid pairs, wherein each pairincludes one nucleic acid encoding a heavy chain candidate variableregion and one nucleic acid encoding a light chain candidate variableregion, for each of a plurality of heavy and light chain variable regionpairs (e.g., obtained from different antibodies binding the same ordifferent antigen(s) of interest);

b) for each of the plurality nucleic acid pairs, making a heterotrimericprotein by:

-   -   (i) producing a first nucleic acid encoding a first polypeptide        chain comprising one of the heavy or light chain candidate        variable domains (V) fused to a first CH1 or CK constant region,        a second variable domain fused to a second CH1 or CK constant        region, and an Fc domain or portion thereof interposed between        the first and second (V—CH1/CK) units);    -   (ii) producing a second nucleic acid encoding a second        polypeptide chain comprising the other of the heavy or light        chain candidate variable domains (V) fused at its C-terminus to        a CH1 or CK constant region selected to be complementary to the        first CH1 or CK constant region of the first polypeptide chain        such that the first and second polypeptides form a CH1-CK        heterodimer in which the heavy and light chain candidate        variable domains form an antigen binding domain;    -   (ii) producing a third nucleic acid encoding a third polypeptide        chain comprising a variable domain fused at its C-terminus to a        CH1 or CK constant region, wherein the CH1 or CK constant region        is selected to be complementary to the second variable domain        and second CH1 or CK constant region of the first polypeptide        chain such that the first and third polypeptides form a CH1-CK        heterodimer in which the second variable domain of the first        polypeptide and the variable domain of the third polypeptide        form an antigen binding domain; and    -   (iii) expressing said nucleic acids encoding the first and        second polypeptide chains in a host cell to produce said first        and second polypeptide chains, respectively; and recovering a        heterodimeric protein; and

c) evaluating the plurality of heterodimeric proteins produced for abiological activity of interest, e,g., an activity disclosed herein. Inone embodiment, the second or the third polypeptide further comprisesand Fc domain or fragment thereof fused to the C-terminus of the CH1 orCK domain (e.g. via a hinge domain or linker). Optionally, theheterotrimeric protein produced represents at least 20%, 25% or 30% ofthe total proteins prior to purification. Optionally the recovering stepin (iii) loading the protein produced onto an affinity purificationsupport, optionally an affinity exchange column, optionally a Protein-Asupport or column, and collecting the heterotrimeric protein; and/orloading the protein produced (e.g., the protein collected followingloading onto an affinity exchange or Protein A column) onto an ionexchange column; and collecting the heterotrimeric fraction.

In the methods for identifying or evaluating candidate variable regions,it will be appreciated that the candidate variable regions can be froman anti-NKp46 antibody or from an antigen that binds an antigen ofinterest. It will also be appreciated that the position of therespective ABDs for the candidate variable region pair and the othervariable region pair can be inverted. For example, in a trimeric proteinthe methods can be modified such that the heavy and light chaincandidate variable domains are formed by the second V region of thefirst polypeptide and the V region of the second polypeptide, and theother variable region pair are formed by the first V region of the firstpolypeptide and the V region of the third polypeptide.

In one embodiment, the second variable domain of the first polypeptideand the variable domain of the third polypeptide form an antigen bindingdomain that binds NKp46. Furthermore, by providing a panel of differentmultispecific protein formats that all can be produced in standard celllines and standardized methods with high yields, yet have differentproperties (e.g. conformational flexibility, spacing between two antigenbinding domains, etc.) that can affect functional activity of theprotein, the protein formats of the disclosure can be used in a panel toscreen proteins configurations or formats to identify the most effectiveconfigurations or formats for a given antigen of interest, orcombination of first and second antigen of interest. Different proteinsformats may access or engage their antigen targets differently.

In one aspect, the present disclosure provides a method for identifyingor evaluating candidate protein configurations for use in aheterodimeric protein, comprising the steps of: producing, separately(e.g. in separate containers), a plurality of multispecific proteins ofthe disclosure, wherein the proteins differ in their domainarrangements, and evaluating the plurality of multispecific proteinsproduced for a biological activity of interest, e.g., an activitydisclosed herein. In one embodiment, the proteins having differentdomain arrangements share antigen binding domains (e.g. the same CDRs orvariable domains) for NKp46 and/or the antigen of interest. In oneembodiment 1, 2, 3, 4, 5, 6, 7 or more different proteins are producedand evaluated. In one embodiment, one or more of (or all of) theproteins are selected from the group of proteins having a domainarrangement disclosed herein, e.g. that of formats F1, F2, F3, F4, F5,F6, F7, F8, F9, F10, F11, F12, F13, F14, F15, F16 and F17. In oneembodiment the proteins are produced according to the methods disclosedherein. Optionally, the plurality of multispecific proteins includes oneprotein with a monomeric Fc domain and one protein with a dimeric Fcdomain.

In one aspect, the present disclosure provides a library of at least 5,10, 20, 30, 50 hetero-multimeric proteins of the disclosure, wherein theproteins share domain arrangements but differ in the amino acid sequenceof the variable domain of one or both of their antigen binding domains.

In one aspect, the present disclosure provides a library of at least 2,3, 4, 5 or 10 hetero-multimeric proteins of the disclosure, wherein theproteins share the amino acid sequence of the variable domain of one orboth of their antigen binding domains, but differ in domainarrangements.

In one aspect of the any of the embodiments herein, recovering amonomeric, heterodimeric or heterotrimer protein can compriseintroducing the protein to a solid phase so as to immobilize theprotein. The immobilized protein can then subsequently be eluted.Generally, the solid support may be any suitable insoluble,functionalized material to which the proteins can be reversiblyattached, either directly or indirectly, allowing them to be separatedfrom unwanted materials, for example, excess reagents, contaminants, andsolvents. Examples of solid supports include, for example,functionalized polymeric materials, e.g., agarose, or its bead formSepharose®, dextran, polystyrene and polypropylene, or mixtures thereof;compact discs comprising microfluidic channel structures; protein arraychips; pipet tips; membranes, e.g., nitrocellulose or PVDF membranes;and microparticles, e.g., paramagnetic or non-paramagnetic beads. Insome embodiments, an affinity medium will be bound to the solid supportand the protein will be indirectly attached to solid support via theaffinity medium. In one aspect, the solid support comprises a protein Aaffinity medium or protein G affinity medium. A “protein A affinitymedium” and a “protein G affinity medium” each refer to a solid phaseonto which is bound a natural or synthetic protein comprising anFc-binding domain of protein A or protein G, respectively, or a mutatedvariant or fragment of an Fc-binding domain of protein A or protein G,respectively, which variant or fragment retains the affinity for anFc-portion of an antibody. Protein A and Protein G are bacterial cellwall proteins that have binding sites for the Fc portion of mammalianIgG. The capacity of these proteins for IgG varies with the species. Ingeneral, IgGs have a higher affinity for Protein G than for Protein A,and Protein G can bind IgG from a wider variety of species. The affinityof various IgG subclasses, especially from mouse and human, for ProteinA varies more than for Protein G. Protein A can, therefore, be used toprepare isotypically pure IgG from some species. When covalentlyattached to a solid matrix, such as cross-linked agarose, these proteinscan be used to capture and purify antigen-protein complexes frombiochemical solutions. Commercially available products include, e.g.,Protein G, A or L bonded to agarose or sepharose beads, for exampleEZview™ Red Protein G Affinity Gel is Protein G covalently bonded to 4%Agarose beads (Sigma Aldrich Co); or POROS® A, G, and CaptureSelect®HPLC columns (Invitrogen Inc.). Affinity capture reagents are alsodescribed, for example, in the Antibody Purification Handbook,Biosciences, publication No. 18-1037-46, Edition AC, the disclosure ofwhich is hereby incorporated by reference).

In one aspect of the any of the embodiments herein, evaluatingmonomeric, heterodimeric or heterotrimeric proteins for a characteristicof interest comprises evaluating the proteins for one or more propertiesselected from the group consisting of: binding to an antigen ofinterest, binding to NKp46, binding to a tumor, viral or bacterialantigen, binding to an FcRn receptor, binding to an Fcγ receptor,Fc-domain mediated effector function(s), agonistic or antagonisticactivity at a polypeptide to which the multimeric proteins binds,ability to modulate the activity (e.g. cause the death of) a cellexpressing the antigen of interest, ability to direct a lymphocyte to acell expressing the antigen of interest, ability to activate alymphocyte in the presence and/or absence of a cell expressing theantigen of interest, NK cell activation, activation of NKp46-expressinglymphocytes (e.g. NK cells) in presence but not in absence of targetcells, lack of activation of NKp46-negative lymphocytes, stability orhalf-life in vitro or in vivo, production yield, purity within acomposition, and susceptibility to aggregate in solution.

In one aspect, the present disclosure provides a method for identifyingor evaluating an anti-NKp46 bispecific protein, comprising the steps of:

(a) providing nucleic acid(s) encoding an anti-NKp46 bispecific proteindescribed herein;

(b) expressing said nucleic acid(s) in a host cell to produce saidprotein, respectively; and recovering said protein; and

(c) evaluating the protein produced for a biological activity ofinterest, e.g., an activity disclosed herein. In one embodiment, aplurality of different anti-NKp46 bispecific proteins are produced andevaluated.

In one embodiment, the step (c) comprises:

(i) testing the ability of the protein to activate effector cells thatexpress NKp46, when incubated with such effector cells in the presenceof target cells (that express antigen of interest). Optionally, step (i)is followed by a step comprising: selecting a protein (e.g., for furtherdevelopment, for use as a medicament) that activates said effectorcells.

In one embodiment, the step (c) comprises:

(i) testing the ability of the protein to activate effector cells thatexpress NKp46, when incubated with such effector cells in the absence oftarget cells (that express antigen of interest). Optionally, step (i) isfollowed by a step comprising: selecting a protein (e.g., for furtherdevelopment, for use as a medicament) that does not substantiallyactivate said effector cells.

In one embodiment, the step (c) comprises:

(i) testing the ability of the protein to activate effector cells thatexpress NKp46, when incubated with such effector cells in the presenceof target cells (that express antigen of interest); and

(ii) testing the ability of the protein to activate effector cells thatexpress NKp46, when incubated with such effector cells in the absence oftarget cells (that express antigen of interest). Optionally, the methodfurther comprises: selecting a protein (e.g., for further development,for use as a medicament) that does not substantially activate saideffector cells when incubated in the absence of target cells, and thatactivates said effector cells when incubated in the presence of targetcells.

In one embodiment, the step (c) comprises:

(i) testing the ability of the polypeptide to induce effector cells thatexpress NKp46 to lyse target cells (that express antigen of interest),when incubated such effector cells in the presence of target cells.Optionally, step (i) is followed by a step comprising: selecting aprotein (e.g., for further development, for use as a medicament) thatinduces effector cells that express NKp46 to lyse the target cells, whenincubated such effector cells in the presence of the target cells.

In one embodiment, the step (c) comprises:

(i) testing the ability of the protein to activate effector cells thatexpress CD16 but do not express NKp46, when incubated with such effectorcells in the presence of target cells. Optionally, step (i) is followedby a step comprising: selecting a protein (e.g., for furtherdevelopment, for use as a medicament) that do not substantially activatesaid effector cells, when incubated with such effector cells in thepresence of target cells.

Uses of Compounds

In one aspect, provided are the use of any of the compounds definedherein for the manufacture of a pharmaceutical preparation for thetreatment or diagnosis of a mammal in need thereof. Provided also arethe use any of the compounds defined above as a medicament or an activecomponent or active substance in a medicament. In a further aspectprovided is a method for preparing a pharmaceutical compositioncontaining a compound as defined above, to provide a solid or a liquidformulation for administration orally, topically, or by injection. Sucha method or process at least comprises the step of mixing the compoundwith a pharmaceutically acceptable carrier.

In one aspect, provided is a method to treat, prevent or more generallyaffect a predefined condition by exerting a certain effect, or detect acertain condition using a multispecific protein described herein, or a(pharmaceutical) composition comprising such.

For example, in one aspect, the invention provides a method of restoringor potentiating the activity of NKp46+NK cells in a patient in needthereof (e.g. a patient having a cancer or a viral or bacterialinfection), comprising the step of administering a multispecific proteindescribed herein to said patient. In one embodiment, the method isdirected at increasing the activity of NKp46+ lymphocytes in patientshaving a disease in which increased lymphocyte (e.g. NK cell) activityis beneficial or which is caused or characterized by insufficient NKcell activity, such as a cancer, or a viral or microbial/bacterialinfection.

The polypeptides described herein can be used to prevent or treatdisorders that can be treated with antibodies, such as cancers, solidand non-solid tumors, hematological malignancies, infections such asviral infections, and inflammatory or autoimmune disorders.

In one embodiment, the antigen of interest (the non-NKp46 antigen) is anantigen expressed on the surface of a malignant cell of a type of cancerselected from the group consisting of: carcinoma, including that of thebladder, head and neck, breast, colon, kidney, liver, lung, ovary,prostate, pancreas, stomach, cervix, thyroid and skin, includingsquamous cell carcinoma; hematopoietic tumors of lymphoid lineage,including leukemia, acute lymphocytic leukemia, acute lymphoblasticleukemia, B-cell lymphoma, T-cell lymphoma, Hodgkins lymphoma,non-Hodgkins lymphoma, hairy cell lymphoma and Burketts lymphoma;hematopoietic tumors of myeloid lineage, including acute and chronicmyelogenous leukemias and promyelocytic leukemia; tumors of mesenchymalorigin, including fibrosarcoma and rhabdomyoscarcoma; other tumors,including neuroblastoma and glioma; tumors of the central and peripheralnervous system, including astrocytoma, neuroblastoma, glioma, andschwannomas; tumors of mesenchymal origin, including fibrosarcoma,rhabdomyoscaroma, and osteosarcoma; and other tumors, includingmelanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroidfollicular cancer and teratocarcinoma, hematopoietic tumors of lymphoidlineage, for example T-cell and B-cell tumors, including but not limitedto T-cell disorders such as T-prolymphocytic leukemia (T-PLL), includingof the small cell and cerebriform cell type; large granular lymphocyteleukemia (LGL) preferably of the T-cell type; Sezary syndrome (SS);Adult T-cell leukemia lymphoma (ATLL); a/d T-NHL hepatosplenic lymphoma;peripheral/post-thymic T cell lymphoma (pleomorphic and immunoblasticsubtypes); angio immunoblastic T-cell lymphoma; angiocentric (nasal)T-cell lymphoma; anaplastic (Ki 1+) large cell lymphoma; intestinalT-cell lymphoma; T-lymphoblastic; and lymphoma/leukaemia (T-Lbly/T-ALL).

In one embodiment, polypeptides described herein can be used to preventor treat a cancer selected from the group consisting of: carcinoma,including that of the bladder, head and neck, breast, colon, kidney,liver, lung, ovary, prostate, pancreas, stomach, cervix, thyroid andskin, including squamous cell carcinoma; hematopoietic tumors oflymphoid lineage, including leukemia, acute lymphocytic leukemia, acutelymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkinslymphoma, non-Hodgkins lymphoma, hairy cell lymphoma and Burkettslymphoma; hematopoietic tumors of myeloid lineage, including acute andchronic myelogenous leukemias and promyelocytic leukemia; tumors ofmesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; othertumors, including neuroblastoma and glioma; tumors of the central andperipheral nervous system, including astrocytoma, neuroblastoma, glioma,and schwannomas; tumors of mesenchymal origin, including fibrosarcoma,rhabdomyoscaroma, and osteosarcoma; and other tumors, includingmelanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroidfollicular cancer and teratocarcinoma. Other exemplary disorders thatcan be treated according to the invention include hematopoietic tumorsof lymphoid lineage, for example T-cell and B-cell tumors, including butnot limited to T-cell disorders such as T-prolymphocytic leukemia(T-PLL), including of the small cell and cerebriform cell type; largegranular lymphocyte leukemia (LGL) preferably of the T-cell type; Sezarysyndrome (SS); Adult T-cell leukemia lymphoma (ATLL); a/d T-NHLhepatosplenic lymphoma; peripheral/post-thymic T cell lymphoma(pleomorphic and immunoblastic subtypes); angio immunoblastic T-celllymphoma; angiocentric (nasal) T-cell lymphoma; anaplastic (Ki 1+) largecell lymphoma; intestinal T-cell lymphoma; T-lymphoblastic; andlymphoma/leukaemia (T-Lbly/T-ALL).

In one example, the tumor antigen is an antigen expressed on the surfaceof a lymphoma cell or a leukemia cell, and the multispecific protein isadministered to, and/or used for the treatment of, an individual havinga lymphoma or a leukemia. Optionally, the tumor antigen is selected fromCD19, CD20, CD22, CD30 or CD33.

In one aspect, the methods of treatment comprise administering to anindividual a multispecific protein described herein in a therapeuticallyeffective amount. A therapeutically effective amount may be any amountthat has a therapeutic effect in a patient having a disease or disorder(or promotes, enhances, and/or induces such an effect in at least asubstantial proportion of patients with the disease or disorder andsubstantially similar characteristics as the patient).

In one embodiment, the multispecific protein described herein may beused in combined treatments with one or more other therapeutic agents,including agents normally utilized for the particular therapeuticpurpose for which the antibody is being administered. The additionaltherapeutic agent will normally be administered in amounts and treatmentregimens typically used for that agent in a monotherapy for theparticular disease or condition being treated. Such therapeutic agentswhen used in the treatment of cancer, include, but are not limited toanti-cancer agents and chemotherapeutic agents; in the treatment ofinfections disease, include, but are not limited to anti-viral agentsand anti-biotics.

In one embodiment, the additional therapeutic agent is an agent capableof inducing ADCC of a cell to which it is bound, e.g. via CD16 expressedby an NK cell. Typically, such protein will have an Fc domain or portionthereof and will exhibit binding to Fcγ receptors (e.g. CD16). In oneembodiment, its ADCC activity will be mediated at least in part by CD16.In one embodiment, the additional therapeutic agent is an antibodyhaving a native or modified human Fc domain, for example a Fc domainfrom a human IgG1 or IgG3 antibody. The term “antibody-dependentcell-mediated cytotoxicity” or “ADCC” is a term well understood in theart, and refers to a cell-mediated reaction in which non-specificcytotoxic cells that express Fc receptors (FcRs) recognize boundantibody on a target cell and subsequently cause lysis of the targetcell. Non-specific cytotoxic cells that mediate ADCC include naturalkiller (NK) cells, macrophages, monocytes, neutrophils, and eosinophils.The term “ADCC-inducing antibody” refers to an antibody thatdemonstrates ADCC as measured by assay(s) known to those of skill in theart. Such activity is typically characterized by the binding of the Fcregion with various FcRs. Without being limited by any particularmechanism, those of skill in the art will recognize that the ability ofan antibody to demonstrate ADCC can be, for example, by virtue of itsubclass (such as IgG1 or IgG3), by mutations introduced into the Fcregion, or by virtue of modifications to the carbohydrate patterns inthe Fc region of the antibody.

Certain modifications to the Fc region of an antibody, as compared to awild type Fc region, are also known by those in the art to enhance ADCCactivity. Combinations with such “ADCC-enhanced” antibodies as theadditional therapeutic agent are particularly advantageous because suchantibodies may induce high activation via CD16, and the multispecificproteins acting via NKp46 will induce NK cell activation and/or targetcell lysis by a complementary mechanism without interfering with CD16pathway utilized by ADCC-enhanced antibodies, and without causingadditional immune-related toxicity. Typical modifications includemodified human IgG1 constant regions comprising at least one amino acidmodification (e.g. substitution, deletions, insertions), and/or alteredtypes of glycosylation, e.g., hypofucosylation. Such modifications canaffect interaction with Fc receptors: FcγRI (CD64), FcγRII (CD32), andFcγRIII (CD 16). FcγRI (CD64), FcγRIIA (CD32A) and FcγRIII (CD 16) areactivating (i.e., immune system enhancing) receptors while FcγRIIB(CD32B) is an inhibiting (i.e., immune system dampening) receptor. Amodification may, for example, increase binding of the Fc domain toFcγRIIIa on effector (e.g. NK) cells and/or decrease binding to FcγRIIB.Examples of modifications are provided in PCT/EP2013/069302 filed 17Sep. 2013, the disclosure of which is incorporated herein by reference.

In some embodiments, the additional therapeutic agent is an antibodycomprising a variant Fc region comprise at least one amino acidmodification (for example, possessing 1, 2, 3, 4, 5, 6, 7, 8, 9, or moreamino acid modifications) in the CH3 domain of the Fc region. In otherembodiments, the antibodies comprising a variant Fc region comprise atleast one amino acid modification (for example, possessing 1, 2, 3, 4,5, 6, 7, 8, 9, or more amino acid modifications) in the CH2 domain ofthe Fc region, which is defined as extending from amino acids 231-341.In some embodiments, antibodies comprise at least two amino acidmodifications (for example, possessing 2, 3, 4, 5, 6, 7, 8, 9, or moreamino acid modifications), wherein at least one such modification is inthe CH3 region and at least one such modification is in the CH2 region.Encompasses also are amino acid modification in the hinge region. In oneembodiment, encompassed are amino acid modification in the CH1 domain ofthe Fc region, which is defined as extending from amino acids 216-230.Any combination of Fc modifications can be made, for example anycombination of different modifications disclosed in United StatesPatents Nos. U.S. Pat. Nos. 7,632,497; 7,521,542; 7,425,619; 7,416,727;7,371,826; 7,355,008; 7,335,742; 7,332,581; 7,183,387; 7,122,637;6,821,505 and 6,737,056; in PCT Publications Nos. WO2011/109400; WO2008/105886; WO 2008/002933; WO 2007/021841; WO 2007/106707; WO06/088494; WO 05/115452; WO 05/110474; WO 04/1032269; WO 00/42072; WO06/088494; WO 07/024249; WO 05/047327; WO 04/099249 and WO 04/063351;and in Lazar et al. (2006) Proc. Nat. Acad. Sci. USA 103(11): 405-410;Presta, L. G. et al. (2002) Biochem. Soc. Trans. 30(4):487-490; Shields,R. L. et al. (2002) J. Biol. Chem. 26; 277(30):26733-26740 and Shields,R. L. et al. (2001) J. Biol. Chem. 276(9):6591-6604).

In some embodiments, the additional therapeutic agent is an antibodycomprising a variant Fc region, wherein the variant Fc region comprisesat least one amino acid modification (for example, possessing 1, 2, 3,4, 5, 6, 7, 8, 9, or more amino acid modifications) relative to awild-type Fc region, such that the molecule has an enhanced effectorfunction relative to a molecule comprising a wild-type Fc region,optionally wherein the variant Fc region comprises a substitution at anyone or more of positions 221, 239, 243, 247, 255, 256, 258, 267, 268,269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294,295, 296, 298, 300, 301, 303, 305, 307, 308, 309, 310, 311, 312, 316,320, 322, 326, 329, 330, 332, 331, 332, 333, 334, 335, 337, 338, 339,340, 359, 360, 370, 373, 376, 378, 392, 396, 399, 402, 404, 416, 419,421, 430, 434, 435, 437, 438 and/or 439. In one embodiment, In someembodiments, the additional therapeutic agent is an antibody comprisinga variant Fc region, wherein the variant Fc region comprises at leastone amino acid modification (for example, possessing 1, 2, 3, 4, 5, 6,7, 8, 9, or more amino acid modifications) relative to a wild-type Fcregion, such that the molecule has an enhanced effector functionrelative to a molecule comprising a wild-type Fc region, optionallywherein the variant Fc region comprises a substitution at any one ormore of positions 239, 298, 330, 332, 333 and/or 334 (e.g. S239D, S298A,A330L, 1332E, E333A and/or K334A substitutions).

In some embodiments, the additional therapeutic agent is an antibodycomprising altered glycosylation patterns that increase Fc receptorbinding ability of antibodies. Such carbohydrate modifications can beaccomplished by, for example, expressing the antibody in a host cellwith altered glycosylation machinery. Cells with altered glycosylationmachinery have been described in the art and can be used as host cellsin which to express recombinant antibodies to thereby produce anantibody with altered glycosylation. See, for example, Shields, R. L. etal. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat.Biotech. 17:176-1, as well as, European Patent No: EP 1,176,195; PCTPublications WO 06/133148; WO 03/035835; WO 99/54342, each of which isincorporated herein by reference in its entirety. In one aspect, theantibodies are hypofucosylated in their constant region. Such antibodiesmay comprise an amino acid alteration or may not comprise an amino acidalteration but be produced or treated under conditions so as to yieldsuch hypofucosylation. In one aspect, an antibody composition comprisesa chimeric, human or humanized antibody described herein, wherein atleast 20, 30, 40, 50, 60, 75, 85, 90, 95% or substantially all of theantibody species in the composition have a constant region comprising acore carbohydrate structure (e.g. complex, hybrid and high mannosestructures) which lacks fucose. In one embodiment, provided is anantibody composition which is free of antibodies comprising a corecarbohydrate structure having fucose. The core carbohydrate willpreferably be a sugar chain at Asn297.

Examples of ADCC-enhanced antibodies include but are not limited to:GA-101 (hypofucosylated anti-CD20), margetuximab (Fc enhancedanti-HER2), mepolizumab, MEDI-551 (Fc engineered anti-CD19),obinutuzumab (glyco-engineered/hypofucosuylated anti-CD20), ocaratuzumab(Fc engineered anti-CD20), XmAb®5574/MOR208 (Fc engineered anti-CD19).

In one example, the additional therapeutic agent (e.g. antibody capableof inducing ADCC) binds a cancer antigen present on a lymphoma or aleukemia cell, e.g. CD19, CD20, CD22, CD30 or CD33, and themultispecific protein and the additional therapeutic agent areadministered to, and/or are used in the treatment of, an individualhaving a lymphoma or a leukemia.

“Combination therapy” embraces the administration of a secondtherapeutic agent (e.g. an ADCC-inducing antibody) and a multispecificprotein described herein as part of a specific treatment regimenintended to provide a beneficial effect from the co-action of thesetherapeutic agents. The beneficial effect of the combination includes,but is not limited to, pharmacokinetic or pharmacodynamic co-actionresulting from the combination of therapeutic agents. Administration ofthese therapeutic agents in combination typically is carried out over adefined time period (usually minutes, hours, days or weeks dependingupon the combination selected). “Combination therapy” generally is notintended to encompass the administration of two or more of thesetherapeutic agents as part of separate monotherapy regimens thatincidentally and arbitrarily result in the combinations of the presentinvention. “Combination therapy” embraces administration of thesetherapeutic agents in a sequential manner, that is, wherein eachtherapeutic agent is administered at a different time, as well asadministration of these therapeutic agents, or at least two of thetherapeutic agents, in a substantially simultaneous manner.Substantially simultaneous administration can be accomplished, forexample, by administering to the subject a single capsule having a fixedratio of each therapeutic agent or in multiple, single capsules for eachof the therapeutic agents. Sequential or substantially simultaneousadministration of each therapeutic agent can be effected by anyappropriate route including, but not limited to, oral routes,intravenous routes, intramuscular routes, and direct absorption throughmucous membrane tissues. The therapeutic agents can be administered bythe same route or by different routes. For example, a first therapeuticagent of the combination selected may be administered by intravenousinjection while the other therapeutic agents of the combination may beadministered orally. Alternatively, for example, both the therapeuticagents may be administered orally or both therapeutic agents may beadministered by intravenous injection. The sequence in which thetherapeutic agents are administered is not narrowly critical.“Combination therapy” also can embrace the administration of thetherapeutic agents as described above in further combination with otherbiologically active ingredients (such as, but not limited to, a secondand different antineoplastic agent) and non-drug therapies (such as, butnot limited to, surgery or radiation treatment).

The multispecific polypeptides can be included in kits. The kits mayoptionally further contain any number of polypeptides and/or othercompounds, e.g., 1, 2, 3, 4, or any other number of multispecificpolypeptide and/or other compounds. It will be appreciated that thisdescription of the contents of the kits is not limiting in any way. Forexample, the kit may contain other types of therapeutic compounds.Optionally, the kits also include instructions for using thepolypeptides, e.g., detailing the herein-described methods.

Also provided are pharmaceutical compositions comprising the compoundsas defined above. A compound may be administered in purified formtogether with a pharmaceutical carrier as a pharmaceutical composition.The form depends on the intended mode of administration and therapeuticor diagnostic application. The pharmaceutical carrier can be anycompatible, nontoxic substance suitable to deliver the compounds to thepatient. Pharmaceutically acceptable carriers are well known in the artand include, for example, aqueous solutions such as (sterile) water orphysiologically buffered saline or other solvents or vehicles such asglycols, glycerol, oils such as olive oil or injectable organic esters,alcohol, fats, waxes, and inert solids A pharmaceutically acceptablecarrier may further contain physiologically acceptable compounds thatact for example to stabilize or to increase the absorption of thecompounds Such physiologically acceptable compounds include, forexample, carbohydrates, such as glucose, sucrose or dextrans,antioxidants, such as ascorbic acid or glutathione, chelating agents,low molecular weight proteins or other stabilizers or excipients Oneskilled in the art would know that the choice of a pharmaceuticallyacceptable carrier, including a physiologically acceptable compound,depends, for example, on the route of administration of the compositionPharmaceutically acceptable adjuvants, buffering agents, dispersingagents, and the like, may also be incorporated into the pharmaceuticalcompositions.

The compounds can be administered parenterally. Preparations of thecompounds for parenteral administration must be sterile. Sterilizationis readily accomplished by filtration through sterile filtrationmembranes, optionally prior to or following lyophilization andreconstitution. The parenteral route for administration of compounds isin accord with known methods, e.g. injection or infusion by intravenous,intraperitoneal, intramuscular, intraarterial, or intralesional routes.The compounds may be administered continuously by infusion or by bolusinjection. A typical composition for intravenous infusion could be madeup to contain 100 to 500 ml of sterile 0.9% NaCl or 5% glucoseoptionally supplemented with a 20% albumin solution and 1 mg to 10 g ofthe compound, depending on the particular type of compound and itsrequired dosing regimen. Methods for preparing parenterallyadministrable compositions are well known in the art.

EXAMPLES Example 1 Generation of Anti-huNKp46 Antibodies

Balb/c mice were immunized with a recombinant human NKp46 extracellulardomain recombinant-Fc protein. Mice received one primo-immunization withan emulsion of 50 μg NKp46 protein and Complete Freund Adjuvant,intraperitoneally, a 2nd immunization with an emulsion of 50 μg NKp46protein and Incomplete Freund Adjuvant, intraperitoneally, and finally aboost with 10 μg NKp46 protein, intravenously. Immune spleen cells werefused 3 days after the boost with X63.Ag8.653 immortalized B cells, andcultured in the presence of irradiated spleen cells.

Primary screen: Supernatant (SN) of growing clones were tested in aprimary screen by flow cytometry using a cell line expressing the humanNKp46 construct at the cell surface. Briefly, for FACS screening, thepresence of reacting antibodies in supernanants was revealed by Goatanti-mouse polyclonal antibody (pAb) labeled with PE.

A selection of antibodies that bound NKp46 were selected, produced andtheir variable regions further evaluated for their activity in thecontext of a bispecific molecule.

Example 2 Identification of a Bispecific Antibody Format that Binds FcRnbut not FcγR for Targeting Effector Cell Receptors

The aim of this experiment was to develop a new bispecific proteinformat that places an Fc domain on a polypeptide together with ananti-NKp46 binding domain and an anti-target antigen binding domain. Thebispecific protein binds to NKp46 monovalently via its anti-NKp46binding domain. The monomeric Fc domain retains at least partial bindingto the human neonatal Fc receptor (FcRn), yet does not substantiallybind human CD16 and/or other human Fcγ receptors. Consequently, thebispecific protein will not induce Fcγ-mediated (e.g. CD16-mediated)target cell lysis.

Example 2-1 Construction and Binding Analysis ofAnti-CD19-IgG1-Fcmono-Anti-CD3

Since no anti-NKp46 bispecific antibody has been produced that couldindicate whether such a protein could be functional, CD3 was used as amodel antigen in place of NKp46 in order to investigate thefunctionality of a new monovalent bispecific protein format prior totargeting NK cells via NKp46.

A bispecific Fc-based on a scFv specific for tumor antigen CD19(anti-CD19 scFv) and a scFV specific for activating receptor CD3 on a Tcell (anti-CD3 scFv) was used to assess FcRn binding and CD19-bindingfunctions of a new monomeric bispecific polypeptide format. The domainarrangement of the final polypeptide is shown in FIG. 2 and is alsoreferred to as the “F1” format (the star in the CH2 domain indicates anoptional N297S mutation, not included in the polypeptide tested here).

A bispecific monomeric Fc-containing polypeptide was constructed basedon an scFv specific for the tumor antigen CD19 (anti-CD19 scFv) and anscFV specific for an activating receptor CD3 on a T cell (anti-CD3scFv). The CH3 domain incorporated the mutations (EU numbering) L351K,T366S, P395V, F405R, T407A and K409Y. The polypeptide has domainsarranged as follows: anti-CD19-CH2-CH3-anti-CD3. DNA sequence coding fora CH3/VH linker peptide having the amino acid sequence STGS was designedin order to insert a specific Sall restriction site at the CH3-VHjunction.

The CH3 domain incorporated the mutations (EU numbering) L351K, T366S,P395V, F405R, T407A and K409Y. The CH2 domain was a wild-type CH2. DNAand amino acid sequences for the monomeric CH2-CH3 Fc portion and theanti-CD19 are shown below.

The light chain and heavy chain DNA and amino acid sequencescorresponding to the anti-CD19 scFv were as follows:

Sequence SEQ ID NO Anti-CD19-VK DNA 113 Anti-CD19-VK amino acid 114Anti-CD19-VH DNA 115 Anti-CD19-VH amino acid 116

The DNA sequences for the monomeric CH2-CH3 Fc portion and finalbispecific IgG1-Fcmono polypeptide (the last K was removed in thatconstruct) is shown in SEQ ID NO: 117. The amino acid sequence is shownin SEQ ID NO: 2. The Anti-CD19-F1-Anti-CD3 complete sequence (matureprotein) is shown in SEQ ID NO: 118.

Cloning and Production of the Recombinant Proteins

Coding sequences were generated by direct synthesis and/or by PCR. PCRwere performed using the PrimeSTAR MAX DNA polymerase (Takara, # R045A)and PCR products were purified from 1% agarose gel using the NucleoSpingel and PCR clean-up kit (Macherey-Nagel, #740609.250). Once purifiedthe PCR product were quantified prior to the In-Fusion ligation reactionperformed as described in the manufacturer's protocol (ClonTech, #ST0345). The plasmids were obtained after a miniprep preparation run onan EVO200 (Tecan) using the Nucleospin 96 plasmid kit (Macherey-Nagel,#740625.4). Plasmids were then sequenced for sequences confirmationbefore to transfecting the CHO cell line.

CHO cells were grown in the CD-CHO medium (Invitrogen) complemented withphenol red and 6 mM GlutaMax. The day before the transfection, cells arecounted and seeded at 175.000 cells/ml. For the transfection, cells(200.000 cells/transfection) are prepared as described in the AMAXA SFcell line kit (AMAXA, # V4XC-2032) and nucleofected using the DS137protocol with the Nucleofector 4D device. All the tranfections wereperformed using 300 ng of verified plasmids. After transfection, cellsare seeded into 24 well plates in pre-warmed culture medium. After 24H,hygromycine B was added in the culture medium (200 μg/ml). Proteinexpression is monitored after one week in culture. Cells expressing theproteins are then sub-cloned to obtain the best producers. Sub-cloningwas performed using 96 flat-bottom well plates in which the cells areseeded at one cell per well into 200 μl of culture medium complementedwith 200 μg/ml of hygromycine B. Cells were left for three weeks beforeto test the clone's productivity.

Recombinant proteins which contain a IgG1-Fc fragment are purified usingProtein-A beads (-rProteinA Sepharose fast flow, GE Healthcare, ref.:17-1279-03). Briefly, cell culture supernatants were concentrated,clarified by centrifugation and injected onto Protein-A columns tocapture the recombinant Fc containing proteins. Proteins were eluted atacidic pH (citric acid 0.1M pH3), immediately neutralized using TRIS-HCLpH8.5 and dialyzed against 1×PBS. Recombinant scFv which contain a “sixhis” tag were purified by affinity chromatography using Cobalt resin.Other recombinant scFv were purified by size exclusion chromatography(SEC).

Example 2-2: Binding Analysis of Anti-CD19-IgG1-Fcmono-Anti-CD3 to B221,JURKAT, HUT78 and CHO Cell Lines

Cells were harvested and stained with the cell supernatant of theanti-CD19-F1-anti-CD3 producing cells during 1 H at 4° C. After twowashes in staining buffer (PBS1X/BSA 0.2%/EDTA 2 mM), cells were stainedfor 30 min at 4° C. with goat anti-human (Fc)-PE antibody (1M0550Beckman Coulter-1/200). After two washes, stainings were acquired on aBD FACS Canto II and analyzed using the FlowJo software.

CD3 and CD19 expression were also controlled by flow cytometry: Cellswere harvested and stained in PBS1X/BSA 0.2%/EDTA 2 mM buffer during 30min at 4° C. using 5p1 of the anti-CD3-APC and 5p1 of the anti-CD19-FITCantibodies. After two washes, stainings were acquired on a BD FACS CantoII and analyzed using the FlowJo software.

The Anti-CD19-F1-Anti-CD3 protein binds to the CD3 cell lines (HUT78 andJURKAT cell lines) and the CD19 cell line (B221 cell line) but not tothe CHO cell line used as a negative control.

Example 2-3 T- and B-Cell Aggregation by Purified Anti-CD19-F1-Anti-CD3

Purified Anti-CD19-F1-Anti-CD3 was tested in a T/B cell aggregationassay to evaluate whether the antibody is functional in bringingtogether CD19 and CD3 expressing cells.

Results are shown in FIG. 4. The top panel shows thatAnti-CD19-F1-Anti-CD3 does not cause aggregation in the presence of B221(CD19) or JURKAT (CD3) cell lines, but it does cause aggregation ofcells when both B221 and JURKAT cells are co-incubated, illustratingthat the bispecific antibody is functional. The lower panel showscontrol without antibody.

Example 2-4 Binding of Bispecific Monomeric Fc Polypeptide to FcRn

Affinity Study by Surface Plasmon Resonance (SPR)

Biacore T100 General Procedure and Reagents

SPR measurements were performed on a Biacore T100 apparatus (Biacore GEHealthcare) at 25° C. In all Biacore experiments Acetate Buffer (50 mMAcetate pH5.6, 150 mM NaCl, 0.1% surfactant p20) and HBS-EP+(Biacore GEHealthcare) served as running buffer and regeneration bufferrespectively. Sensorgrams were analyzed with Biacore T100 Evaluationsoftware. Recombinant mouse FcRn was purchase from R&D Systems.

Immobilization of FcRn

Recombinant FcRn proteins were immobilized covalently to carboxyl groupsin the dextran layer on a Sensor Chip CM5. The chip surface wasactivated with EDC/NHS (N-ethyl-N′-(3-dimethylaminopropyl)carbodiimidehydrochloride and N-hydroxysuccinimide (Biacore GEHealthcare)). FcRn proteins were diluted to 10 μg/ml in coupling buffer(10 mM acetate, pH 5.6) and injected until the appropriateimmobilization level was reached (i.e. 2500 RU). Deactivation of theremaining activated groups was performed using 100 mM ethanolamine pH 8(Biacore GE Healthcare).

Affinity Study

Monovalent affinity study was done following the Single Cycle Kinetic(SCK) protocol. Five serial dilutions of soluble analytes (antibodiesand bi-specific molecules) ranging from 41.5 to 660 nM were injectedover the FcRn (without regeneration) and allowed to dissociate for 10min before regeneration. For each analyte, the entire sensorgram wasfitted using the 1:1 SCK binding model.

Results

Anti-CD19-F1-Anti-CD3 having its CH2-CH3 domains placed between twoantigen binding domains, here two scFv, was evaluated to assess whethersuch bispecific monomeric Fc protein could retain binding to FcRn andthereby have improved in vivo half-lives compared to conventionbispecific antibodies. Results showed that FcRn binding was retained,the model suggesting 1:1 ratio (1 FcRn for each monomeric Fc) instead of2:1 ration (2 FcRn for each antibody) for a regular IgG. Results areshown in FIG. 5.

Affinity was evaluated using SPR, in comparison to a chimeric fulllength antibody having human IgG1 constant regions. Results are shown inFIG. 5. The monomeric Fc retained significant monomeric binding to FcRn(monomeric Fc: affinity of KD=194 nM; full length antibody with bivalentbinding: avidity of KD=15.4 nM).

Example 3 Construction of Anti-CD19×Anti-NKp46 Bispecific Monomeric FcDomain Polypeptides

It was unknown what activating receptors on NK cells would contribute tolysis of target cells, and since anti-NKp46 antibodies may block NKp46,whether cytotoxicity could be mediated by NKp46 triggering. Weinvestigated whether the bispecific protein format could induce NKp46triggering, and moreover without inducing NKp46 agonism in the absenceof target cells, which could lead to inappropriate NK activation distantfrom the target and/or decreased overall activity toward target cells.

A new bispecific protein format was developed as a single chain proteinwhich binds to FcRn but not FcγR. Additionally, multimeric proteins thatcomprise two or three polypeptide chains, wherein the Fc domain remainsmonomeric, were developed that are compatible for use with antibodyvariable regions that do not maintain binding to their target whenconverted to scFv format. The latter formats can be used convenientlyfor antibody screening; by incorporating at least one binding region asa F(ab) structure, any anti-target (e.g. anti-tumor) antibody variableregion can be directly expressed in a bispecific construct as the F(ab)format within the bispecific protein and tested, irrespective of whetherthe antibody would retain binding as an scFv, thereby simplifyingscreening and enhancing the number of antibodies available. Theseformats in which the Fc domain remains monomeric have the advantage ofmaintaining maximum conformational flexibility which may permit optimalbinding to NKp46 or target antigens.

Different constructs were made for use in the preparation of abispecific antibodies using the variable domains DNA and amino acidsequences from the scFv specific for tumor antigen CD19 described inExample 2-1, and different variable regions from antibodies specific forthe NKp46 receptor identified in Example 1. A construct was also madeusing as anti-NKp46 the variable regions from existing antibody Bab281(mIgG1, available commercially from Beckman Coulter, Inc. (Brea, Calif.,USA) (see also Pessino et al, J. Exp. Med, 1998, 188 (5): 953-960 andSivori et al, Eur J Immunol, 1999. 29:1656-1666) specific for the NKp46receptor.

For the Fc domain to remain monomeric in single chain polypeptides ormultimers in which only one chain had an Fc domain, CH3-CH3 dimerizationwas prevented through two different strategies: (1) through the use ofCH3 domain incorporating the mutations (EU numbering) L351 K, T366S,P395V, F405R, T407A and K409Y; or (2) through the use of a tandem CH3domain in which the tandem CH3 domains separated by a flexible linkerassociated with one another, in turn preventing interchain CH3-CH3dimerization. The DNA and amino acid sequences for the monomeric CH2-CH3Fc portion with point mutations were as in Example 2-1. The DNA andamino acid sequences for the monomeric CH2-CH3-linker-CH3 Fc portionwith tandem CH3 domains is shown in FIGS. 6A-6D.

The light chain and heavy chain DNA and amino acid sequences for theanti-CD19 scFv were as in Example 2-1. Proteins were cloned, producedand purified as in Example 2-1. Shown below are the light chain andheavy chain DNA and amino acid sequences for anti-NKp46 scFv.

TABLE 1 Amino acid sequences of different anti-NKp46 scFv scFv anti-NKp46 scFV sequence (VHVK)/- stop NKp46-1STGSQVQLQQSGPELVKPGASVKMSCKASGYTFTDYVINWGKQRSGQGLEWIGEIYPGSGTNYYNEKFKAKATLTADKSSNIAYMQLSSLTSEDSAVYFCARRGRYGLYAMDYWGQGTSVTVSSVEGGSGGSGGSGGSGGVDDIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTINNLEQEDIATYFCQQGNTRPWTFGGGTKLEIK- (SEQ ID NO: 119) NKp46-2STGSEVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYITYSGSTSYNPSLESRISITRDTSTNQFFLQLNSVTTEDTATYYCARGGYYGSSWGVFAYWGQGTLVTVSAVEGGSGGSGGSGGSGGVDDIQMTQSPASLSASVGETVTITCRVSENIYSYLAWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDEGSYYCQHHYGTPWTEGGGTKLEIK- (SEQ ID NO: 120) NKp46-3STGSEVQLQQSGPELVKPGASVKISCKTSGYTFTEYTMHWVKQSHGKSLEWIGGISPNIGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRGGSFDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLELK- (SEQ ID NO: 121) NKp46-4STGSQVQLQQSAVELARPGASVKMSCKASGYTFTSFTMHWVKQRPGQGLEWIGYINPSSGYTEYNQKFKDKTTLTADKSSSTAYMQLDSLTSDDSAVYYCVRGSSRGFDYWGQGTLVTVSAVEGGSGGSGGSGGSGGVDDIQMIQSPASLSVSVGETVTITCRASENIYSNLAWFQQKQGKSPQLLVYAATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGIYYCQHFWGTPRTFGGGTKLEIK- (SEQ ID NO: 122) NKp46-6STGSQVQLQQPGSVLVRPGASVKLSCKASGYTFTSSWMHWAKQRPGQGLEWIGHIHPNSGISNYNEKFKGKATLTVDTSSSTAYVDLSSLTSEDSAVYYCARGGRFDDWGAGTTVTVSSVEGGSGGSGGSGGSGGVDDIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFLMYTEGGGTKLEIK- (SEQ ID NO: 123) NKp46-9STGSDVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYITYSGSTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARCWDYALYAMDCWGQGTSVTVSSVEGGSGGSGGSGGSGGVDDIQMTQSPASLSASVGETVTITCRTSENIYSYLAWCQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTHFSLKINSLQPEDFGIYYCQHHYDTPLTFGAGTKLELK- (SEQ ID NO: 124) Bab281STGSQIQLVQSGPELQKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTNTGEPTYAEEFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARDYLYYFDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDNIVMTQSPKSMSMSVGERVTLTCKASENVVTYVSWYQQKPEQSPKLLIYGASNRYTGVPDRFTGSGSATDFTLTISSVQAEDLADYHCGQGYSYPYTFGGGTKLEIK- (SEQ ID NO: 125)

TABLE 2 DNA sequences corresponding to the different anti-NKp46 scFvscFv anti-NKp46 scFV sequences NKp46-1 SEQ ID NO: 126 NKp46-2 SEQ ID NO:127 NKp46-3 SEQ ID NO: 128 NKp46-4 SEQ ID NO: 129 NKp46-6 SEQ ID NO: 130NKp46-9 SEQ ID NO: 131 Bab281 SEQ ID NO: 132

Format 1 (F1) (Anti-CD19-IgG1-Fcmono-Anti-NKp46 (scFv))

The domain structure of Format 1 (F1) is shown in FIG. 6A. A bispecificFc-containing polypeptide was constructed based on an scFv specific forthe tumor antigen CD19 (anti-CD19 scFv) and an scFV specific for theNKp46 receptor. The polypeptide is a single chain polypeptide havingdomains arranged (N- to C-terminal) as follows:

(VK—VH)^(anti-CD19)-CH2-CH3-(VH—VK)^(anti-NKp46)

A DNA sequence coding for a CH3/VH linker peptide having the amino acidsequence STGS was designed in order to insert a specific Sallrestriction site at the CH3-VH junction. The domain arrangement of thefinal polypeptide in shown in FIG. 2 (star in the CH2 domain indicatesan optional N297S mutation), where the anti-CD3 scFv is replaced by ananti-NKp46 scFv. The (VK—VH) units include a linker between the VH andVK domains. Proteins were cloned, produced and purified as in Example2-1. The amino acid sequences of the bispecific polypeptides (completesequence (mature protein)) are shown in the corresponding SEQ ID NOSlisted in the table 3 below.

TABLE 3 Sequence SEQ ID NO CD19-F1-NKp46-1 133 CD19-F1-NKp46-2 134CD19-F1-NKp46-3 135 CD19-F1-NKp46-4 136 CD19-F1-NKp46-6 137CD19-F1-NKp46-9 138 CD19-F1-Bab281 139

Format 2 (F2): CD19-F2-NKp46-3

The domain structure of F2 polypeptides is shown in FIG. 6A. The DNA andamino acid sequences for the monomeric CH2-CH3 Fc portion were as inExample 2-1 containing CH3 domain mutations (the mutations (EUnumbering) L351K, T366S, P395V, F405R, T407A and K409Y. The heterodimeris made up of:

a first (central) polypeptide chain having domains arranged as follows(N- to C-terminal): (VK—VH)^(anti-CD19)-CH2-CH3-VH^(anti-NKp46)-CH1  (1)

and

a second polypeptide chain having domains arranged as follows (N- toC-terminal): VK^(anti-NKp46)CK.  (2)

The (VK—VH) unit was made up of a VH domain, a linker and a VK unit(i.e. an scFv). As with other formats of the bispecific polypeptides,the DNA sequence coded for a CH3/VH linker peptide having the amino acidsequence STGS designed in order to insert a specific Sall restrictionsite at the CH3-VH junction. Proteins were cloned, produced and purifiedas in Example 2-1. The amino acid sequences for the first and secondchains of the F2 protein are shown in SEQ ID NO: 140 and 141.

Format 3 (F3): CD19-F3-NKp46-3

The domain structure of F3 polypeptides is shown in FIG. 6A. The DNA andamino acid sequences for the CH2-CH3 Fc portion comprised a tandem CH3domain in which the two CH3 domains on the same polypeptide chainassociated with one another, thereby preventing dimerization betweendifferent bispecific proteins.

The single chain polypeptide has domains arranged (N- to C-terminal) asfollows:

(VK—VH)^(anti-CD19)-CH2-CH3-CH3-(VH—VK)^(anti-NKp46)

The (VK—VH) units were made up of a VH domain, a linker and a VK unit(scFv). Proteins were cloned, produced and purified as in Example 2-1.Bispecific protein was purified from cell culture supernatant byaffinity chromatography using prot-A beads and analysed and purified bySEC. The protein showed a high production yield of 3.4 mg/L and with asimple SEC profile. The amino acid sequence for the F3 protein is shownin SEQ ID NO: 142.

Format 4 (F4): CD19-F4-NKp46-3

The domain structure of F4 polypeptides is shown in FIG. 6A. The DNA andamino acid sequences for the CH2-CH3 Fc portion comprised a tandem CH3domain as in Format F3, however additionally comprising a N297S mutationto prevent N-linked glycosylation and abolish FcγR binding. Proteinswere cloned, produced and purified as in Example 2-1. Bispecificproteins was purified from cell culture supernatant by affinitychromatography using prot-A beads and analysed and purified by SEC. Theprotein showed a good production yield of 1 mg/L and with a simple SECprofile. The amino acid sequence for the F4 protein with NKp46-3variable domains is shown in SEQ ID NO: 143.

Format 8 (F8)

The domain structure of F8 polypeptides is shown in FIG. 6B. The DNA andamino acid sequences for the monomeric CH2-CH3 Fc portion were as inFormat F2 containing CH3 domain mutations (the mutations (EU numbering)L351K, T366S, P395V, F405R, T407A and K409Y, as well as a N297S mutationto prevent N-linked glycosylation and abolish FcγR binding. Threevariants of F8 proteins were produced: (a) cysteine residues in thehinge region left intact (wild-type, referred to as F8A), (b) cysteineresidues in the hinge region replaced by serine residues (F8B), and (c)a linker sequence GGGSS replacing residues DKTHTCPPCP in the hinge(F8C). Variants F8B and F8C provided advantages in production byavoiding formation of homodimers of the central chain. The heterotrimeris made up of;

a first (central) polypeptide chain having domains arranged as follows(N- to C-terminal): VH^(anti-CD19)-CH1-CH2 CH3-VH^(anti-NKp46)-CK  (1)

and

a second polypeptide chain having domains arranged as follows (N- toC-terminal): VK^(anti-NKp46)-CH1  (2)

and

a third polypeptide chain having domains arranged as follows (N- toC-terminal): VK^(anti-CD19)-CK  (3)

Proteins were cloned, produced and purified as in Example 2-1.Bispecific proteins was purified from cell culture supernatant byaffinity chromatography using prot-A beads and analysed and purified bySEC. The protein showed a high production yield of 3.7 mg/L (F8C) andwith a simple SEC profile. The amino acid sequences of the three chainsof the F8 protein (C variant) with NKp46-3 variable regions are shown inSEQ ID NOS: 144, 145 and 146.

Format 9 (F9): CD19-F9-NKp46-3

The F9 polypeptide is a trimeric polypeptide having a centralpolypeptide chain and two polypeptide chains each of which associatewith the central chain via CH1-CK dimerization. The domain structure ofthe trimeric F9 protein is shown in FIG. 6B, wherein the bonds betweenthe CH1 and CK domains are interchain disulfide bonds. The two antigenbinding domains have a F(ab) structure permitting the use of antibodiesirrespective of whether they remain functional in scFv format. The DNAand amino acid sequences for the CH2-CH3 Fc portion comprised a tandemCH3 domain as in Format F4 and a CH2 domain comprising a N297Ssubstitution. Three variants of F9 proteins were produced: (a) cysteineresidues in the hinge region left intact (wild-type, referred to asF9A), (b) cysteine residues in the hinge region replaced by serineresidues (F9B), and (c) a linker sequence GGGSS replacing residuesDKTHTCPPCP in the hinge (F9C). Variants F9B and F9C provided advantagesin production by avoiding formation of homodimers of the central chain.The heterotrimer is made up of:

a first (central) polypeptide chain having domains arranged as follows(N- to C-terminal):VH^(anti-CD19)-CH1-CH2-CH3-CH3-VH^(anti-NKp46)-CK  (1)

and

a second polypeptide chain having domains arranged as follows (N- toC-terminal): VK^(anti-NKp46)-CH1  (2)

and

a third polypeptide chain having domains arranged as follows (N- toC-terminal): VK^(anti-CD19)-CK  (3)

Proteins were cloned, produced and purified as in Example 2-1.Bispecific proteins was purified from cell culture supernatant byaffinity chromatography using prot-A beads and analysed and purified bySEC. The protein showed a high production yield of 8.7 mg/L (F9A) and3.0 mg/L (F9B), and with a simple SEC profile.

The amino acid sequences of the three chains of the F9 protein variantF9A are shown in the SEQ ID NOS: 147, 148 and 149. The amino acidsequences of the three chains of the F9 protein variant F9B are shown inthe SEQ ID NOS: 150, 151 and 152. The amino acid sequences of the threechains of the F9 protein variant F9C are shown in the SEQ ID NOS: 153,154 and 155.

Format 10 (F10): CD19-F10-NKp46-3

The F10 polypeptide is a dimeric protein having a central polypeptidechain and a second polypeptide chain which associates with the centralchain via CH1-CK dimerization. The domain structure of the dimeric F10proteins is shown in FIG. 6B wherein the bonds between the CH1 and CKdomains are interchain disulfide bonds. One of the two antigen bindingdomains has a Fab structure, and the other is a scFv. The DNA and aminoacid sequences for the CH2-CH3 Fc portion comprised a tandem CH3 domainas in Format F4 and a CH2 domain with a N297S substitution.Additionally, three variants of F10 proteins were produced: (a) cysteineresidues in the hinge region left intact (wild-type, referred to asF10A), (b) cysteine residues in the hinge region replaced by serineresidues (F10B, and (c) a linker sequence GGGSS replacing residuesDKTHTCPPCP in the hinge (F10C). Variants F10B an F10C providedadvantages in production by avoiding formation of homodimers of thecentral chain. The (VK—VH) unit was made up of a VH domain, a linker anda VK unit (scFv). The heterodimer is made up of:

a first (central) polypeptide chain having domains arranged as follows(N- to C-terminal):VH^(anti-CD19)-CH1-CH2-CH3-CH3-(VH—VK)^(anti-NKp46)  (1)

and

a second polypeptide chain having domains arranged as follows (N- toC-terminal): VK^(anti-CD19)-CK.  (2)

Proteins were cloned, produced and purified as in Example 2-1.Bispecific proteins was purified from cell culture supernatant byaffinity chromatography using prot-A beads and analysed and purified bySEC. The protein showed a good production yield of 2 mg/L (F10A) andwith a simple SEC profile. The amino acid sequences of the two chains ofthe F10A protein variant are shown in the SEQ ID NOS: 156 (second chain)and 157 (first chain). The amino acid sequences of the two chains of theF10B protein variant are shown in the SEQ ID NOS: 158 (second chain) and159 (first chain). The amino acid sequences of the two chains of theF10C protein variant are shown in the SEQ ID NOS: 160 (second chain) and161 (first chain).

Format 11 (F11): CD19-F11-NKp46-3

The domain structure of F11 polypeptides is shown in FIG. 6C. Theheterodimeric protein is similar to F10 but the structures of theantigen binding domains are reversed. One of the two antigen bindingdomains has a Fab-like structure, and the other is a scFv. Theheterodimer is made up of

a first (central) polypeptide chain having domains arranged as follows(N- to C-terminal): (VK—VH)^(anti-CD19)-CH2-CH3-CH3-VH^(anti-NKp46)CK  (1)

and

a second polypeptide chain having domains arranged as follows (N- toC-terminal): VK^(anti-NKp46)-CH1.  (2)

Proteins were cloned, produced and purified as in Example 2-1.Bispecific proteins was purified from cell culture supernatant byaffinity chromatography using prot-A beads and analysed and purified bySEC. The protein showed a good production yield of 2 mg/L and with asimple SEC profile. The amino acid sequences of the two chains of theF11 protein are shown in SEQ ID NO: 162 (chain 1) and SEQ ID NO: 163(chain 2).

Format 12 (F12): CD19-F12-NKp46-3

The domain structure of the dimeric F12 polypeptides is shown in FIG.6C, wherein the bonds between the CH1 and CK domains are disulfidebonds. The heterodimeric protein is similar to F11 but the CH1 and CKdomains within the F(ab) structure are inversed. The heterodimer is madeup of:

a first (central) polypeptide chain having domains arranged as follows(N- to C-terminal):(VK—VH)^(anti-CD19)-CH2-CH3-CH3-VH^(anti-NKp46)-CH1  (1)

and

a second polypeptide chain having domains arranged as follows (N- toC-terminal): VK^(anti-NKp46)-CK.  (2)

Proteins were cloned, produced and purified as in Example 2-1.Bispecific proteins was purified from cell culture supernatant byaffinity chromatography using prot-A beads and analysed and purified bySEC. The protein showed a good production yield of 2.8 mg/L and with asimple SEC profile. The DNA and amino acid sequences for the F12 proteinare shown below. The amino acid sequences of the two chains of the F12protein are shown in SEQ ID NO: 164 (chain 1) and SEQ ID NO: 165 (chain2).

Format 17 (F17): CD19-F17-NKp46-3

The domain structure of the trimeric F17 polypeptides is shown in FIG.6C, wherein the bonds between the CH1 and CK domains are disulfidebonds. The heterodimeric protein is similar to F9 but the VH and VKdomains, and the CH1 and CK, domains within the C-terminal F(ab)structure are each respectively inversed with their partner. Theheterotrimer is made up of:

a first (central) polypeptide chain having domains arranged as follows(N- to C-terminal):VH^(anti-CD19)-CH1-CH2-CH3-CH3-VK^(anti-NKp46)_-CH1  (1)

and

a second polypeptide chain having domains arranged as follows (N- toC-terminal): VH^(anti-NKp46)-CK  (2)

and

a third polypeptide chain having domains arranged as follows (N- toC-terminal): VK^(anti-CD19)-CK  (3)

Additionally, three variants of F17 proteins were produced: (a) cysteineresidues in the hinge region left intact (wild-type, referred to asF17A), (b) cysteine residues in the hinge region replaced by serineresidues (F10B, and (c) a linker sequence GGGSS replacing residuesDKTHTCPPCP in the hinge (F17C). Proteins were cloned, produced andpurified as in Example 2-1. The amino acid sequences of the three chainsof the F17B protein are shown in SEQ ID NOS: 166, 167 and 168.

Example 4 Bispecific NKp46 Antibody Formats with Dimeric Fc Domains

New protein constructions with dimeric Fc domains were developed thatshare advantages of the monomeric Fc domain proteins of Example 3 butbind to FcRn with greater affinity, but which also have low orsubstantially lack of binding to FcγR. The polypeptide formats weretested to investigate the functionality of heterodimeric proteinscomprising a central chain with a (VH—(CH1/CK)-CH2-CH3-) unit or a(VK—(CH1 or CK)-CH2-CH3-) unit. One of both of the CH3 domains will thenbe fused, optionally via intervening amino acid sequences or domains, toa variable domain(s) (a single variable domain that associates with avariable domain on a separated polypeptide chain, a tandem variabledomain (e.g., an scFv), or a single variable domain that is capable ofbinding antigen as a single variable domain. The two chains thenassociate by CH1-CK dimerization to form disulfide linked dimers, or ifassociated with a third chain, to form trimers. Members of this familyof formats may have less conformational flexibility compared to nativeantibodies or other bispecific constructs.

Different constructs were made for use in the preparation of abispecific antibody using the variable domains DNA and amino acidsequences derived from the scFv specific for tumor antigen CD19described in Example 2-1 and different variable regions from antibodiesspecific for NKp46 identified in Example 1. Proteins were cloned,produced and purified as in Example 2-1. Domains structures are shown inFIGS. 6A-6D.

Format 5 (F5): CD19-F5-NKp46-3

The domain structure of the trimeric F5 polypeptide is shown in FIG. 6D,wherein the interchain bonds between hinge domains (indicated in thefigures between CH1/CK and CH2 domains on a chain) and interchain bondsbetween the CH1 and CK domains are interchain disulfide bonds. Theheterotrimer is made up of:

a first (central) polypeptide chain having domains arranged as follows(N- to C-terminal): VH^(anti-CD19)-CH1-CH2-CH3-VH^(anti-NKp46)-CK  (1)

and

a second polypeptide chain having domains arranged as follows (N- toC-terminal): VK^(anti-CD19)-CK—CH2-CH3  (2)

and

a third polypeptide chain having domains arranged as follows (N- toC-terminal): VK^(anti-NKp46)-CH1  (3)

Proteins were cloned, produced and purified as in Example 2-1.Bispecific proteins was purified from cell culture supernatant byaffinity chromatography using prot-A beads and analysed and purified bySEC. The protein showed a high production yield of 37 mg/L and with asimple SEC profile. The amino acid sequences of the three polypeptidechains are shown in SEQ ID NOS 169 (second chain), 170 (first chain) and171 (third chain).

Format 6 (F6): CD19-F6-NKp46-3

The domain structure of heterotrimeric F6 polypeptides is shown in FIG.6D. The F6 protein is the same as F5, but with a N297S substitution toavoid N-linked glycosylation. Proteins were cloned, produced andpurified as in Example 2-1. Bispecific proteins was purified from cellculture supernatant by affinity chromatography using prot-A beads andanalysed and purified by SEC. The protein showed a high production yieldof 12 mg/L and with a simple SEC profile. The amino acid sequences ofthe three polypeptide chains are shown in SEQ ID NOS: 172 (secondchain), 173 (first chain) and 174 (third chain).

Format 7 (F7): CD19-F7-NKp46-3

The domain structure of heterotrimeric F7 polypeptides is shown in FIG.6D. The F7 protein is the same as F6, but with cysteine to serinesubstitutions in the CH1 and CK domains that are linked at theirC-termini to the Fc domains, to prevent formation of a minor populationof dimeric species of the central chain with the VK^(anti-NKp46)-CH1chain. Proteins were cloned, produced and purified as in Example 2-1.Bispecific proteins was purified from cell culture supernatant byaffinity chromatography using prot-A beads and analysed and purified bySEC. The protein showed a high production yield of 11 mg/L and with asimple SEC profile. The amino acid sequences of the three polypeptidechains are shown in SEQ ID NOS: 175 (second chain), 176 (first chain)and 177 (third chain).

Format 13 (F13): CD19-F13-NKp46-3

The domain structure of the dimeric F13 polypeptide is shown in FIG. 6D,wherein the interchain bonds between hinge domains (indicated betweenCH1/CK and CH2 domains on a chain) and interchain bonds between the CH1and CK domains are interchain disulfide bonds. The heterodimer is madeup of:

a first (central) polypeptide chain having domains arranged as follows(N- to C-terminal): VH^(anti-CD19)-CH1-CH2-CH3-(VH—VK)^(anti-NKp46)  (1)

and

a second polypeptide chain having domains arranged as follows (N- toC-terminal): VK^(anti-CD19)-CK—CH2-CH3.  (2)

The (VH—VK) unit was made up of a VH domain, a linker and a VK unit(scFv).

Proteins were cloned, produced and purified as in Example 2-1.Bispecific proteins was purified from cell culture supernatant byaffinity chromatography using prot-A beads and analysed and purified bySEC. The protein showed a high production yield of 6.4 mg/L and with asimple SEC profile. The amino acid sequences of the two polypeptidechains are shown in SEQ ID NOS: 178 (second chain) and 179 (firstchain).

Format 14 (F14): CD19-F14-NKp46-3

The domain structure of the dimeric F14 polypeptide is shown in FIG. 6E.The F14 polypeptide is a dimeric polypeptide which shares the structureof the F13 format, but instead of a wild-type Fc domain (CH2-CH3), theF14 has CH2 domain mutations N297S to abolish N-linked glycosylation.Proteins were cloned, produced and purified as in Example 2-1.Bispecific proteins was purified from cell culture supernatant byaffinity chromatography using prot-A beads and analysed and purified bySEC. The protein showed a high production yield of 2.4 mg/L and with asimple SEC profile. The amino acid sequences of the two polypeptidechains are shown in SEQ ID NOS: 180 (second chain) and 181 (firstchain).

Format 15 (F15): CD19-F15-NKp46-3

The domain structure of the trimeric F15 polypeptides is shown in FIG.6E. The F15 polypeptide is a dimeric polypeptide which shares thestructure of the F6 format, but differs by inversion of the N-terminalVH—CH1 and VK—CK units between the central and second chains. Proteinswere cloned, produced and purified as in Example 2-1. Bispecificproteins was purified from cell culture supernatant by affinitychromatography using prot-A beads and analysed and purified by SEC. Theprotein showed a good production yield of 0.9 mg/L and with a simple SECprofile. The amino acid sequences of the three polypeptide chains areshown in SEQ ID NOS: 182 (second chain), 183 (first chain) and 184(third chain).

Format 16 (F16): CD19-F16-NKp46-3

The domain structure of the trimeric F16 polypeptide is shown in FIG.6E. The F16 polypeptide is a dimeric polypeptide which shares thestructure of the F6 format, but differs by inversion of the C-terminalVH—CK and VK—CH1 units between the central and second chains. Proteinswere cloned, produced and purified as in Example 2-1. The amino acidsequences of the three polypeptide chains are shown in SEQ ID NOS: 185(second chain), 186 (first chain) and 187 (third chain).

Example 5 NKp46 Binding Affinity by Bispecific Proteins by SurfacePlasmon Resonance (SPR)

Biacore T100 General Procedure and Reagents

SPR measurements were performed on a Biacore T100 apparatus (Biacore GEHealthcare) at 25° C. In all Biacore experiments HBS-EP+(Biacore GEHealthcare) and NaOH 10 mM served as running buffer and regenerationbuffer respectively. Sensorgrams were analyzed with Biacore T100Evaluation software. Protein-A was purchase from (GE Healthcare). HumanNKp46 recombinant proteins were cloned, produced and purified at InnatePharma.

Immobilization of Protein-A

Protein-A proteins were immobilized covalently to carboxyl groups in thedextran layer on a Sensor Chip CMS. The chip surface was activated withEDC/NHS (N-ethyl-N′-(3-dimethylaminopropyl) carbodiimidehydrochlorideand N-hydroxysuccinimide (Biacore GE Healthcare)). Protein-A was dilutedto 10 μg/ml in coupling buffer (10 mM acetate, pH 5.6) and injecteduntil the appropriate immobilization level was reached (i.e. 2000 RU).Deactivation of the remaining activated groups was performed using 100mM ethanolamine pH 8 (Biacore GE Healthcare).

Binding Study

The bispecific proteins were first tested in Format F1 described inExample 2 having different anti-NKp46 variable regions from NKp46-1,NKp46-2, NKp46-3 or NKp46-4 antibodies. Antibodies were next tested asdifferent formats F3, F4, F5, F6, F9, F10, F11, F13, F14 having theanti-NKp46 variable regions from the NKp46-3 antibody, and compared tothe NKp46-3 antibody as a full-length human IgG1.

Bispecific proteins at 1 μg/mL were captured onto Protein-A chip andrecombinant human NKp46 proteins were injected at 5 μg/mL over capturedbispecific antibodies. For blank subtraction, cycles were performedagain replacing NKp46 proteins with running buffer.

The Bab281 antibody was separately tested for binding to NKp46 by SPR,and additionally by flow cytometry using a cell line expressing thehuman NKp46 construct at the cell surface. For FACS screening, thepresence of reacting antibodies in supernanants was revealed by Goatanti-mouse polyclonal antibody (pAb) labeled with PE. SPC and FACSresults showed that the Bab281 based antibody did not bind the NKp46cell line or NKp46-Fc proteins. Bab281 lost binding to its target whenpresented in the bispecific format.

Affinity Study

Monovalent affinity study was done following a regular Capture-Kineticprotocol recommended by the manufacturer (Biacore GE Healthcare kineticwizard). Seven serial dilutions of human NKp46 recombinant proteins,ranging from 6.25 to 400 nM were sequentially injected over the capturedBi-Specific antibodies and allowed to dissociate for 10 min beforeregeneration. The entire sensorgram sets were fitted using the 1:1kinetic binding model.

Results

SPR showed that the bispecific polypeptides of format F1 having theNKp46-1, 2, 3 and 4 scFv binding domains bound to NKp46, while otherbispecific polypeptides having the scFv of other anti-NK46 antibodiesdid not retain NKp46 binding. The binding domains that did not retainbinding in monomeric bispecific format initially bound to NKp46 but lostbinding upon conversion to the bispecific format. All of the bispecificpolypeptides of formats F1, F2 F3, F4, F5, F6, F9, F10, F11, F13, F14retained binding to NKp46 when using the NKp46-3 variable regions.

FIG. 7A shows representative superimposed sensorgrams showing the rawdata curves, sample (CD19-F1-NKp46-1) and blank (Buffer), which wereused to generate each subtracted sensorgrams of FIG. 7B. Subtractedsensorgrams were obtained by subtracting the blank sensorgram to thesample sensorgram. Sensorgrams were aligned to zero in the y and x axisat the capture step injection start before blank subtraction.

FIG. 7B shows representative superimposed substracted sensorgramsshowing the binding of CD19-F1-NKp46-1 recombinant proteins to thecaptured bispecific monomeric polypeptide. Sensorgrams were aligned tozero in the y and x axis at the sample step injection start.

Monovalent affinities and kinetic association and dissociation rateconstants are shown below in the table 3 below.

TABLE 3 Bispecific mAb ka (1/Ms) kd (1/s) KD (M) CD19-F1-Bab281 n/a n/an/a (loss of binding) CD19-F1-NKp46-1  1.23E+05 0.001337 1.09E−08 CD19-F1-NKp46-2  1.62E+05 0.001445 8.93E−09  CD19-F1-NKp46-3  7.05E+046.44E−04 9.14E−09  CD19-F1-NKp46-4  1.35E+05 6.53E−04 4.85E−09 CD19-F3-NKp46-3 3.905E+5 0.01117 28E−09 CD19-F4-NKp46-3 3.678E+5 0.0110030E−09 CD19-F5-NKp46-3 7.555E+4 0.00510 67E−09 CD19-F6-NKp46-3 7.934E+40.00503 63E−09 CD19-F9A-NKp46-3 2.070E+5 0.00669 32E−09CD19-F10A-NKp46-3 2.607E+5 0.00754 29E−09 CD19-F11A-NKp46-3 3.388E+50.01044 30E−09 CD19-F13-NKp46-3 8.300E+4 0.00565 68E−09 CD19-F14-NKp46-38.826E+4 0.00546 62E−09 NKp46-3 IgG1 2.224E+5 0.00433 20E−09

Example 6 Engagement of NK Cells Against Daudi Tumor Target withFc-Containing NKp46×CD19 Bispecific Protein

Bispecific antibodies having a monomeric Fc domain and a domainarrangement according to the single chain F1 or dimeric F2 formatsdescribed in Example 3, and a NKp46 binding region based on NKp46-1,NKp46-2, NKp46-3 or NKp46-4 were tested for functional ability to directNK cells to lyse CD19-positive tumor target cells (Daudi, a wellcharacterized B lymphoblast cell line). The F2 proteins additionallyincluded NKp46-9 variable regions which lost binding to NKp46 in thescFv format but which retained binding in the F(ab)-like format of F2.

Briefly, the cytolytic activity of each of (a) resting human NK cells,and (b) human NK cell line KHYG-1 transfected with human NKp46, wasassessed in a classical 4-h ⁵¹Cr-release assay in U-bottom 96 wellplates. Daudi cells were labelled with ⁵¹Cr (50 μCi (1.85 MBq)/1×10⁶cells), then mixed with KHYG-1 transfected with hNKp46 at aneffector/target ratio equal to 50 for KHYG-1, and 10 (for F1 proteins)or 8.8 (for F2 proteins) for resting NK cells, in the presence ofmonomeric bi-specific antibodies at different concentrations. Afterbrief centrifugation and 4 hours of incubation at 37° C., samples ofsupernatant were removed and transferred into a LumaPlate (Perkin ElmerLife Sciences, Boston, Mass.), and ⁵¹Cr release was measured with aTopCount NXT beta detector (PerkinElmer Life Sciences, Boston, Mass.).All experimental conditions were analyzed in triplicate, and thepercentage of specific lysis was determined as follows: 100×(mean cpmexperimental release—mean cpm spontaneous release)/(mean cpm totalrelease—mean cpm spontaneous release). Percentage of total release isobtained by lysis of target cells with 2% Triton X100 (Sigma) andspontaneous release corresponds to target cells in medium (withouteffectors or Abs).

Results

In the KHYG-1 hNKp46 NK experimental model, each bi-specific antibodyNKp46-1, NKp46-2, NKp46-3, NKp46-4 or NKp46-9 induced specific lysis ofDaudi cells by human KHYG-1 hNKp46 NK cell line compared to negativecontrols (Human IgG1 isotype control (IC) and CD19/CD3 bi-specificantibodies), thereby showing that these antibodies induce Daudi targetcell lysis by KHYG-1 hNKp46 through CD19/NKp46 cross-linking.

When resting NK cells were used as effectors, each bi-specific antibodyNKp46-1, NKp46-2, NKp46-3, NKp46-4 or NKp46-9 again induced specificlysis of Daudi cells by human NK cells compared to negative control(Human IgG1 isotype control (IC) antibody), thereby showing that theseantibodies induce Daudi target cell lysis by human NK cells throughCD19/NKp46 cross-linking. Rituximab (RTX, chimeric IgG1) was used as apositive control of ADCC (Antibody-Dependent Cell Cytotoxicity) byresting human NK cells. The maximal response obtained with RTX (at 10μg/ml in this assay) was 21.6% specific lysis illustrating that thebispecific antibodies have high target cell lysis activity. Results forexperiments with resting NK cells are shown in FIG. 8A for the singlechain F1 proteins and 8B for the dimeric F2 proteins.

Example 7 Comparison with Full Length Anti-NKp46 mAbs and DepletingAnti-Tumor mAbs: Only NKp46×CD19 Bispecific Proteins PreventNon-Specific NK Activation

These studies aimed to investigate whether bispecific antibodies canmediate NKp46-mediated NK activation toward cancer target cells withouttriggering non-specific NK cell activation.

NKp46×CD19 bispecific proteins having an arrangement according to the F2format described in Example 3 with anti-NKp46 variable domains fromNKp46-1, NKp46-2, NKp46-3, NKp46-4 or NKp46-9 were compared to:

(a) full-length monospecific anti-NKp46 antibodies (NKp46-3 as humanIgG1), and

(b) the anti-CD19 antibody as a full-length human IgG1 as ADCC inducingantibody control comparator.

The experiments further included as controls: rituximab, an anti-CD20ADCC inducing antibody control for a target antigen with high expressionlevels; anti-CD52 antibody alemtuzumab, a human IgG1, binds CD52 targetpresent on both targets and NK cells; and negative control isotypecontrol therapeutic antibody (a human IgG1 that does not bind a targetpresent on the target cells (HUG1-IC).

The different proteins were tested for functional effect on NK cellactivation in the presence of CD19-positive tumor target cells (Daudicells), in the presence of CD19-negative, CD16-positive target cells(HUT78 T-lymphoma cells), and in the absence of target cells.

Briefly, NK activation was tested by assessing CD69 and CD107 expressionon NK cells by flow cytometry. The assay was carried out in 96 U wellplates in completed RPMI, 150 μL final/well. Effector cells were freshNK cells purified from donors. Target cells were Daudi (CD19-positive),HUT78 (CD19-negative) or K562 (NK activation control cell line). Inaddition to K562 positive control, three conditions were tested, asfollows:

-   -   NK cell alone    -   NK cells vs Daudi (CD19+)    -   NK cells vs HUT78 (CD19−)

Effector:Target (E:T) ratio was 2.5:1 (50 000 E: 20 000 T), with anantibody dilution range starting to 10 μg/mL with 1/4 dilution (n=8concentrations). Antibodies, target cells and effector cells were mixed;spun 1 min at 300 g; incubated 4 h at 37° C.; spun 3 min at 500 g;washed twice with Staining Buffer (SB); added 50 μL of staining Ab mix;incubated 30 min at 300 g; washed twice with SB resuspended pellet withCellFix; stored overnight at 4° C.; and fluorescence revealed with CantoII (HTS).

Results

1. NK Cells Alone

Results are shown in FIG. 9A. In the absence of target-antigenexpressing cells, none of the bispecific anti-NKp46×anti-CD19 antibody(including each of the NKp46-1, NKp46-2, NKp46-3, NKp46-4 and NKp46-9variable regions) activated NK cells as assessed by CD69 or CD107expression. Full-length anti-CD19 also did not activate NK cells.However, the full-length anti-NKp46 antibodies caused detectableactivation of NK cells. Alemtuzumab also induced activation of NK cells,at a very high level. Isotype control antibody did not induceactivation.

2. NK Cells Vs Daudi (CD19+)

Results are shown in FIG. 9B. In the presence of target-antigenexpressing cells, each of the bispecific anti-NKp46×anti-CD19 antibodies(including each of the NKp46-1, NKp46-2, NKp46-3, NKp46-4 and NKp46-9binding domains) activated NK cells. Full-length anti-CD19 showed atbest only very low activation of NK cells. Neither full-lengthanti-NKp46 antibodies or alemtuzmab showed substantial increase inactivation beyond what was observed in presence of NK cells alone. FIG.9 shows full-length anti-NKp46 antibodies showed a similar level ofbaseline activation observed in presence of NK cells alone. Alemtuzumabalso induced activation of NK cells a similar level of activationobserved in presence of NK cells alone, and at higher antibodyconcentrations in this setting (ET 2.5:1) the activation was greaterthan with the bispecific anti-NKp46×anti-CD19 antibody. Isotype controlantibody did not induce activation.

3. NK Cells Vs HUT78 (CD19-)

Results are shown in FIG. 9C. In the presence of target-antigen-negativeHUT78 cells, none of the bispecific anti-NKp46×anti-CD19 antibody(including each of the NKp46-1, NKp46-2, NKp46-3, NKp46-4 and NKp46-9variable regions) activated NK cells. However, the full-lengthanti-NKp46 antibodies and alemtuzumab caused detectable activation of NKcells at a similar level observed in presence of NK cells alone. Isotypecontrol antibody did not induce activation.

In conclusion, the bispecific anti-NKp46 proteins are able to activateNK cells in a target-cell specific manner, unlike full-lengthmonospecific anti-NKp46 antibodies and full-length antibodies ofdepleting IgG isotypes which also activate NK cells in the absence oftarget cells. The NK cell activation achieved with anti-NKp46 bispecificproteins was higher than that observed with full length anti-CD19 IgG1antibodies.

Example 8 Comparative Efficacy with Depleting Anti-Tumor mAbs:NKp46×CD19 Bispecific Proteins at Low ET Ratio

These studies aimed to investigate whether bispecific antibodies canmediate NKp46-mediated NK cell activation toward cancer target cells atlower effector:target ratios. The ET ratio used in this Example was 1:1which is believed to be closer to the setting that would be encounteredin vivo than the 2.5:1 ET ratio used in Example 7 or the 10:1 ET ratioof Example 6.

NKp46×CD19 bispecific proteins having an arrangement according to the F2format described in Example 3 with anti-NKp46 variable domains fromNKp46-1, NKp46-2, NKp46-3, NKp46-4 or NKp46-9 were compared to:

(a) full-length monospecific anti-NKp46 antibodies (NKp46-3 as humanIgG1), and

(b) the anti-CD19 antibody as a full-length human IgG1 as ADCC inducingantibody control comparator.

The experiments further included as controls: rituximab (an anti-CD20ADCC inducing antibody control for a target antigen with high expressionlevels); anti-CD52 antibody alemtuzumab (a human IgG1, binds CD52 targetpresent on both targets and NK cells), and negative control isotypecontrol therapeutic antibody (a human IgG1 that does not bind a targetpresent on the target cells (HUG1-IC). The different proteins weretested for functional effect on NK cell activation as assessed by CD69or CD107 expression in the presence of CD19-positive tumor target cells(Daudi cells), in the presence of CD19-negative, CD16-positive targetcells (HUT78 T-lymphoma cells), and in the absence of target cells. Theexperiments were carried out as in Example 7 except that the ET ratiowas 1:1.

Results

Results are shown in FIG. 10 (10A: CD107 and 10B: CD69). In the presenceof target-antigen expressing cells, each of the bispecificanti-NKp46×anti-CD19 antibody (including each of the NKp46-1, NKp46-2,NKp46-3, NKp46-4 and NKp46-9 variable regions) activated NK cells in thepresence of Daudi cells.

The activation induced by bispecific anti-NKp46×anti-CD19 antibody inthe presence of Daudi cells was far more potent than the full-lengthhuman IgG1 anti-CD19 antibody as ADCC inducing antibody which had lowactivity in this setting. Furthermore, in this low E:T ratio setting theactivation induced by bispecific anti-NKp46×anti-CD19 antibody was aspotent as anti-CD20 antibody rituximab, with a difference being observedonly at the highest concentrations that were 10 fold higher thanconcentrations in which differences were observed at the 2.5:1 ET ratio.

In the absence of target cells or in the in the presence of targetantigen-negative HUT78 cells, full-length anti-NKp46 antibodies andalemtuzumab showed a similar level of baseline activation observed inthe presence of Daudi cells. Anti-NKp46×anti-CD19 antibody did notactivate NK cells in presence of HUT78 cells.

In conclusion, the bispecific anti-NKp46 proteins are able to activateNK cells in a target-cell specific manner and at lower effector:targetratio are more effective in mediating NK cell activation thattraditional human IgG1 antibodies.

Example 9 Mechanism of Action Studies

NKp46×CD19 bispecific proteins having an arrangement according to theF2, F3, F5 or F6 formats described in Examples 3 or 4 with anti-NKp46variable domains from NKp46-3 were compared to rituximab (anti-CD20 ADCCinducing antibody), and a human IgG1 isotype control antibody forfunctional ability to direct CD16-/NKp46+NK cell lines to lyseCD19-positive tumor target cells.

Briefly, the cytolytic activity of the CD16-/NKp46+ human NK cell lineKHYG-1 was assessed in a classical 4-h ⁵¹Cr-release assay in U-bottom 96well plates. Daudi or B221 cells were labelled with ⁵¹Cr (50 μCi (1.85MBq)/1×10⁶ cells), then mixed with KHYG-1 at an effector/target ratioequal to 50:1, in the presence of test antibodies at dilution rangestarting from 10⁻⁷ mol/L with 1/5 dilution (n=8 concentrations)

After brief centrifugation and 4 hours of incubation at 37° C., 50 μL ofsupernatant were removed and transferred into a LumaPlate (Perkin ElmerLife Sciences, Boston, Mass.), and ⁵¹Cr release was measured with aTopCount NXT beta detector (PerkinElmer Life Sciences, Boston, Mass.).All experimental conditions were analyzed in triplicate, and thepercentage of specific lysis was determined as follows: 100×(mean cpmexperimental release—mean cpm spontaneous release)/(mean cpm totalrelease—mean cpm spontaneous release). Percentage of total release isobtained by lysis of target cells with 2% Triton X100 (Sigma) andspontaneous release corresponds to target cells in medium (withouteffectors or Abs).

Results

Results are shown in FIG. 11A (KHYG-1 vs Daudi) and 11B (KHYG-1 vsB221). In the KHYG-1 hNKp46 NK experimental model, each NKp46×CD19bispecific protein (Format F2, F3, F5 and F6) induced specific lysis ofDaudi or B221 cells by human KHYG-1 hNKp46 NK cell line, while rituximaband human IgG1 isotype control (IC) antibodies did not.

Example 10 Binding of Different Bispecific Formats to FcRn

Affinity of different antibody formats for human FcRn was studied bySurface Plasmon Resonance (SPR) by immobilizing recombinant FcRnproteins covalently to carboxyl groups in the dextran layer on a SensorChip CM5, as described in Example 2-6.

A chimeric full length anti-CD19 antibody having human IgG1 constantregions and NKp46×CD19 bispecific proteins having an arrangementaccording to the F3, F4, F5, F6, F9, F10, F11, F13 or F14 formatsdescribed in Examples 3 or 4 with anti-NKp46 variable domains fromNKp46-3 (NKp46-2 for F2) were tested; for each analyte, the entiresensorgram was fitted using the steady state or 1:1 SCK binding model.

Results are shown in Table 4 below. The bispecific proteins havingdimeric Fc domains (formats F5, F6, F13, F14) bound to FcRn withaffinity similar to that of the full-length IgG1 antibody. Thebispecific proteins with monomeric Fc domains (F3, F4, F9, F10, F11)also displayed binding to FcRn, however with lower affinity that thebispecific proteins having dimeric Fc domains.

TABLE 4 Antibody/Bispecific SPR method KD nM Human IgG1/K Anti-CD19SCK/Two state reaction 7.8 CD19-F5-NKp46-3 SCK/Two state reaction 2.6CD19-F6-NKp46-3 SCK/Two state reaction 6.0 CD19-F13-NKp46-3 SCK/Twostate reaction 15.2 CD19-F14-NKp46-3 SCK/Two state reaction 14.0CD19-F3-NKp46-3 Steady State 474.4 CD19-F4-NKp46-3 Steady State 711.7CD19-F9A-NKp46-3 Steady State 858.5 CD19-F10A-NKp46-3 Steady State 432.8CD19-F11-NKp46-3 Steady State 595.5

Example 11 Binding to FcγR

Anti-CD19-F1-Anti-NKp46 having its CH2-CH3 domains placed between twoantigen binding domains, here two scFv, was evaluated to assess whethersuch bispecific monomeric Fc protein could retain binding to Fcγreceptors.

Human IgG1 antibodies and CD19/NKp46-1 bi-specific antibodies wereimmobilized onto a CM5 chip. Recombinant FcγRs (cynomolgus monkey andhuman CD64, CD32a, CD32b, and CD16) were cloned, produced and purifiedat Innate Pharma. FIG. 18 shows superimposed sensorgrams showing thebinding of Macaca fascicularis recombinant FcgRs (upper panels; CyCD64,CyCD32a, CYCD32b, CyCD16) and of Human recombinant FcgRs (lower panels;HuCD64, HuCD32a, HuCD32b, HuCD16a) to the immobilized human IgG1 control(grey) and CD19/NKp46-1 bi-specific antibody (black). Sensorgrams werealigned to zero in the y and x axis at the sample injection start.

FIG. 18 shows that while full length wild type human IgG1 bound to allcynomolgus and human Fcγ receptors, the CD19/NKp46-1 bi-specificantibodies did not bind to any of the receptors

Example 12 Epitope Mapping of Anti-NKp46 Antibodies

A. Competition Assays

Competition assays were conducted by Surface Plasmon Resonance (SPRaccording to the methods described below.

Biacore T100 General Procedure and Reagents

SPR measurements were performed on a Biacore T100 apparatus (Biacore GEHealthcare) at 25° C. In all Biacore experiments HBS-EP+(Biacore GEHealthcare) and NaOH 10 mM NaCl 500 mM served as running buffer andregeneration buffer respectively. Sensorgrams were analyzed with BiacoreT100 Evaluation software. Anti-6xHis tag antibody was purchased fromQIAGEN. Human 6xHis tagged NKp46 recombinant proteins (NKp46-His) werecloned, produced and purified at Innate Pharma.

Immobilization of Anti-6xHis Tag Antibodies

Anti-His antibodies were immobilized covalently to carboxyl groups inthe dextran layer on a Sensor Chip CM5. The chip surface was activatedwith EDC/NHS (N-ethyl-N′-(3-dimethylaminopropyl)carbodiimidehydrochloride and N-hydroxysuccinimide (Biacore GEHealthcare)). Protein-A and Anti-His antibodies were diluted to 10 μg/mlin coupling buffer (10 mM acetate, pH 5.6) and injected until theappropriate immobilization level was reached (i.e. 2000 to 2500 RU).Deactivation of the remaining activated groups was performed using 100mM ethanolamine pH 8 (Biacore GE Healthcare).

Competition Study

Parental regular human IgG1 chimeric antibodies having NKp46 bindingregion corresponding to NKp46-1, NKp46-2, NKp46-3 or NKp46-4 were usedfor the competition study which has been performed using an Anti-6xHistag antibody chip.

Bispecific antibodies having NKp46 binding region based on NKp46-1,NKp46-2, NKp46-3 or NKp46-4 at 1 μg/mL were captured onto Protein-A chipand recombinant human NKp46 proteins were injected at 5 μg/mL togetherwith a second test bispecific antibody of the NKp46-1, NKp46-2, NKp46-3or NKp46-4 group.

None of NKp46-1, NKp46-2, NKp46-3 or NKp46-4 competed with one anotherfor binding to NKp46, these antibodies each representing a differentepitope.

B. Binding to NKp46 Mutants

In order to define the epitopes of anti NKp46 antibodies, we designedNKp46 mutants defined by one, two or three substitutions of amino acidsexposed at the molecular surface over the 2 domains of NKp46. Thisapproach led to the generation of 42 mutants transfected in Hek-293Tcells, as shown in the table below. The targeted amino acid mutations inthe table 5 below are shown both using numbering of SEQ ID NO: 1 (alsocorresponding to the numbering used in Jaron-Mendelson et al. (2012) J.Immunol. 88(12):6165-74.

TABLE 5 Substitution (Numbering according to Jaron- Mutant Mendelson andSEQ ID NO 1) 1 P40A K43S Q44A 2 K41S E42A E119A 3 P86A D87A 4 N89A R91A5 K80A K82A  5 bis E34A T46A 6 R101A V102A 7 N52A Y53A 8 V56A P75A E76A9 R77A I78A 10 S97A I99A 10 bis Q59A H61A 11 L66A V69A 12 E108A 13 N111AL112A 14 D114A 15 T125A R145S D147A 16 S127A Y143A 17 H129A K139A 18K170A V172A 19 I135A S136A 19 bis T182A R185A 20 R160A 21 K207A 22 M152AR166A 23 N195A N196A Stalk1 D213A I214A T217A Stalk2 F226A T233A Stalk3L236A T240A Supp1 F30A W32A Supp2 F62A F67A Supp3 E63A Q95A Supp4 R71AK73A Supp5 Y84A Supp6 E104A L105A Supp7 Y121A Y194A Supp8 P132A E133ASupp9 S151A Y168A Supp10 S162A H163A Supp11 E174A P176A Supp12 P179AH184A Supp13 R189A E204A P205A

Generation of Mutants

NKp46 mutants were generated by PCR. The sequences amplified were run onagarose gel and purified using the Macherey Nagel PCR Clean-Up GelExtraction kit (reference 740609). The two or three purified PCRproducts generated for each mutant were then ligated into an expressionvector, with the ClonTech InFusion system. The vectors containing themutated sequences were prepared as Miniprep and sequenced. Aftersequencing, the vectors containing the mutated sequences were preparedas Midiprep using the Promega PureYield™ Plasmid Midiprep System.HEK293T cells were grown in DMEM medium (Invitrogen), transfected withvectors using Invitrogen's Lipofectamine 2000 and incubated at 37° C. ina CO2 incubator for 24 hours prior to testing for transgene expression.

Flow Cytometry Analysis of Anti-NKp46 Binding to the HEK293T TransfectedCells

All the anti-NKp46 antibodies were tested for their binding to eachmutant by flow cytometry. A first experiment was performed to determineantibodies that lose their binding to one or several mutants at oneconcentration (10 μg/ml). To confirm a loss of binding, titration ofantibodies was done on antibodies for which binding seemed to beaffected by the NKp46 mutations (1−0,1−0,01−0,001 μg/ml).

Results

Results are shown in FIGS. 12 to 17. Antibody NKp46-1 had decreasedbinding to the mutant 2 (having a mutation at residues K41, E42 andE119, as shown in FIG. 12A (NKp46wild-type) compared to 12B (mutant 2).Similarly, NKp46-1 also had decreased binding to the supplementarymutant Supp7 (having a mutation at residues Y121 and Y194, as shown inFIG. 13A (NKp46 wild-type) compared to 13B (mutant Supp7).

Antibody NKp46-3 had decreased binding to the mutant 19 (having amutation at residues 1135, and S136, as shown in FIG. 15A (NKp46wild-type) compared to 15B (mutant 19). Similarly, NKp46-1 also haddecreased binding to the supplementary mutant Supp8 (having a mutationat residues P132 and E133, as shown in FIG. 14A (NKp46 wild-type)compared to 14B (mutant Supp8).

Antibody NKp46-4 had decreased binding to the mutant 6 (having amutation at residues R101, and V102, as shown in FIG. 16A (NKp46wild-type) compared to 16B (mutant 6). Similarly, NKp46-1 also haddecreased binding to the supplementary mutant Supp6 (having a mutationat residues E104 and L105, as shown in FIG. 17A (NKp46 wild-type)compared to 17B (mutant Supp6).

In this study, we identified epitopes for anti-NKp46 antibodies(NKp46-1, NKp46-3 and NKp46-4). Epitopes of NKp46-4, NKp46-3 and NKp46-1are on NKp46 D1 domain, D2 domain and D1/D2 junction, respectively.R101, V102, E104 and L105 are essential residues for NKp46-4 binding anddefined a part of NKp46-4 epitope. The epitope of NKp46-1 epitopeincludes K41, E42, E119, Y121 and Y194 residues. The epitope of NKp46-3includes P132, E133, 1135, and S136 residues.

Example 13 Improved Product Profile and Yield of Different BispecificFormats Compared to Existing Formats

Blinatumomab and two bispecific antibodies having NKp46 and CD19 bindingregions based on F1 to F17 formats and NKp46-3, and blinatumomab,respectively were cloned and produced under format 6 (F6), DART and BITEformats following the same protocol and using the same expressionsystem. F6, DART and BITE bispecific proteins were purified from cellculture supernatant by affinity chromatography using prot-A beads for F6or Ni-NTA beads for DART and BITE. Purified proteins were furtheranalysed and purified by SEC (FIG. 19-A). BITE and DART showed a verylow production yield compared to F6 and have a very complex SEC profile.As shown in FIG. 19-B (arrows), DART and BITE are barely detectable bySDS-PAGE after Coomassie staining in the expected SEC fractions (3 and 4for BITE and 4 and 5 for DART), whereas F6 format showed clear andsimple SEC and SDS-PAGE profiles with a major peak (fraction 3)containing the multimeric bispecific proteins. The major peak for the F6format corresponded to about 30% of the total proteins. Theseobservations are also true for F1 to F17 proteins (data not shown)indicating that the Fc domain (or Fc-derive domain) present in thoseformats facilitate the production and improve the quality and solubilityof bispecific proteins.

Moreover, the Fc domains present in proteins F1 to F17 have theadvantage of being adapted to affinity chromatography without the needfor incorporation of peptide tags that will thereafter remain present asan unwanted part of a therapeutic product, such as in the case of BiTeand DART antibodies which cannot be purified by protein A. F1 to F17antibodies are all bound by protein A. Table 6 below shows productivityof different formats.

TABLE 6 Final SDS PAGE « productivity » Format SEC Reduced Non Reducedyield F3 2 peaks ✓ ✓ 3.4 mg/L F4 2 peaks ✓ ✓ 1 mg/L F5 ✓ ✓ ✓ 37 mg/L F6✓ ✓ ✓ 12 mg/L F7 ✓ ✓ ✓ 11 mg/L F8C ✓ ✓ ✓ 3.7 mg/L F9A ✓ ✓ ✓ 8.7 mg/L F9B✓ ✓ ✓ 3.0 mg/L F10A ✓ ✓ ✓ 2.0 mg/L F11 ✓ ✓ ✓ 2.0 mg/L F12 ✓ ✓ ✓ 2.8 mg/LF13 ✓ ✓ ✓ 6.4 mg/L F14 ✓ ✓ ✓ 2.4 mg/L F15 ✓ ✓ ✓ 0.9 mg/L BiTe — — — —DART — — — —

All headings and sub-headings are used herein for convenience only andshould not be construed as limiting the invention in any way. Anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context. Recitation ofranges of values herein are merely intended to serve as a shorthandmethod of referring individually to each separate value falling withinthe range, unless otherwise indicated herein, and each separate value isincorporated into the specification as if it were individually recitedherein. Unless otherwise stated, all exact values provided herein arerepresentative of corresponding approximate values (e. g., all exactexemplary values provided with respect to a particular factor ormeasurement can be considered to also provide a correspondingapproximate measurement, modified by “about,” where appropriate). Allmethods described herein can be performed in any suitable order unlessotherwise indicated herein or otherwise clearly contradicted by context.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise indicated. No language in the specification should beconstrued as indicating any element is essential to the practice of theinvention unless as much is explicitly stated.

The description herein of any aspect or embodiment of the inventionusing terms such as reference to an element or elements is intended toprovide support for a similar aspect or embodiment of the invention that“consists of,” “consists essentially of” or “substantially comprises”that particular element or elements, unless otherwise stated or clearlycontradicted by context (e.g., a composition described herein ascomprising a particular element should be understood as also describinga composition consisting of that element, unless otherwise stated orclearly contradicted by context).

This invention includes all modifications and equivalents of the subjectmatter recited in the aspects or claims presented herein to the maximumextent permitted by applicable law.

All publications and patent applications cited in this specification areherein incorporated by reference in their entireties as if eachindividual publication or patent application were specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1. An isolated multispecific protein comprising a first antigen bindingdomain and a second antigen binding domain, wherein one of the first orsecond antigen binding domains binds to a human NKp46 polypeptide andthe other binds an antigen of interest, wherein the multispecificprotein binds the NKp46 polypeptide monovalently, and wherein themultispecific protein is capable of directing an NKp46-expressing NKcell to lyse a target cell expressing the antigen of interest. 2-49.(canceled)